Vh4 codon signature for multiple sclerosis

ABSTRACT

The present invention provides for the diagnosis and prediction of multiple sclerosis (MS) in subject utilizing a unique a codon signature in VH4 expressing B cells that has now been associated with MS and not with any other autoimmune disease.

This application is a continuation of U.S. application Ser. No.12/509,093, filed Jul. 24, 2009, which claims benefit of U.S.Provisional Application Ser. No. 61/083,429, filed Jul. 24, 2008. Theentire contents of each of the above referenced disclosures are herebyincorporated by reference.

This invention was made with government support under grant no. NS 40993awarded by the National Institutes of Health. The government has certainrights in the invention.

Pursuant to 37 C.F.R. 1.821(c), a sequence listing is submitted herewithas an ASCII compliant text file named “UTSDP2107USC2.txt”, created onMar. 7, 2013 and having a size of ˜27 KB. The content of theaforementioned file is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to fields of pathology, immunology andmolecular biology. More particularly, the present invention relates to aVH4 peptide signature in VH-expressing B cells that predicts anddiagnoses multiple sclerosis.

2. Description of Related Art

B cells have historically been implicated in the pathogenesis ofMultiple Sclerosis (MS) since elevated CNS immunoglobulins andoligoclonal bands were first described in MS patients in the 1940s(Kabat et al. 1950; Kabat et al., 1948). Additional evidence of B cellinvolvement in MS pathogenesis includes the presence of B cells in MSlesions (Raine et al., 1999), and presence of B cells trafficking intothe CNS during lesion development (Esiri, 1977) that intensifies withdisease duration (Ozawa et al., 1994). Furthermore, the ratio of B cellsto monocytes is stable over the disease course, but those patients witha prevalence of B cells tend to have a more expeditious diseaseprogression than those with monocyte predominance (Cepok et al., 2001).Antibodies in conjunction with complement have also been identified inMS lesions co-localized with disintegrating myelin, suggesting apotential causative role played by these immune elements in lesiondevelopment (Genain et al., 1999; Storch and Lassmann, 1997).

The inventor and others have shown that clonal expansion of B cellsoccurs in both cerebrospinal fluid (CSF) (Colombo et al., 2000; Monsonet al., 2005; Owens et al., 2003; Qin et al., 1998; Ritchie et al.,2004) and lesion sites (Baranzini et al., 1999; Owens et al., 1998) ofMS patients. Clones at different stages of affinity maturation can befound in the CSF, suggesting that expansion is local (Monson et al.,2005). This finding is further substantiated by evidence that bothectopic germinal centers (Magliozzi et al., 2004; Serafini et al., 2004;Uccelli et al., 2005) and centroblasts, specialized B cells only foundin germinal centers (Corcione et al., 2004), can be detectable in theCNS of MS patients. Characterization of clonally expanded B cells fromthe CSF of MS patients (MSCSF) demonstrated that the antibodies these Bcells express are often self-reactive towards antigens found in thebrain (Qin et al., 1998; Lambracht-Washington et al., 2007).

Data from others have established that the VH4-expressing B cellpopulation in particular harbors autoreactive B cells in both healthycontrols (Koelsch et al., 2007) and patients with systemic lupuserythematosus (SLE) (Pugh-Bernard et al., 2001). For example, B cellsutilizing the VH4-34 gene (formerly known as VH4-21) are oftenautoreactive towards sugars on blood cells and either undergo negativeselection in the adult repertoire (Koelsch et al., 2007; Pascual andCapra, 1992), or class switch to the rare IgD isotype rather than IgG,presumably to dampen the response of these autoreactive cells in healthyindividuals (Koelsch et al., 2007).

Autoreactive B cells from the CSF and brain lesions (i.e., CNS) of MSpatients are not suppressed, but instead undergo extensive clonalexpansion (Colombo et al., 2000; Monson et al., 2005; Owens et al.,2003; Qin et al., 1998; Ritchie et al., 2004; Owens et al., 1998;Lambracht-Washington et al., 2007; Harp et al., 2007; Owens et al.,2007). This observation may indicate that regulatory mechanisms in theCNS of these patients may be more flexible than they are in theperiphery. However, the inventor's previous analysis of antibodyrepertoires from MS patient derived CSF B cells indicated thatregulation of this population, as it relates to germinal centerselection, is preserved (Harp et al., 2007). The exception to thisfinding is that some, but not all, individual clonal populations appearto be dysregulated, as evidenced by lack of mutational targeting (Monsonet al., 2005). A number of independent laboratories have documented thatVH4-expressing B cells are overrepresented in the CSF (Colombo et al.,2000; Monson et al., 2005; Owens et al., 2003; Qin et al., 1998; Ritchieet al., 2004) and brain lesions (Baranzini et al., 1999; Owens et al.,1998) of MS patients. This finding was inconspicuous, however, sinceclonally expanding and autoreactive B cells from the CSF of MS patientscould be found utilizing variable genes from any of the heavy (andlight) chain families (Buluwela and Rabbitts, 1988; Humphries et al.,1988; Kodaira et al. 1986; Lee et al., 1987; Shen et al., 1987).

SUMMARY OF THE INVENTION

Thus, in accordance with the present invention, there is provided amethod for identifying a human subject having or at risk of developingmultiple sclerosis (MS) comprising assessing the VH4 structure of aVH4-expressing B-cell from said subject, wherein the presence of a codonsignature associated with MS identifies said subject as having or atrisk of developing MS. The codon signature may comprise a mutation atcodon 31B, 56 and/or 81, or mutations at each of 31B, 56 and 81. Thecodon signature may further comprise mutations at one or more of codons32, 40, 52, 57, 60 and 89, such as mutations at each of codons 31B, 32,40, 56, 57, 60, 81 and 89. The codon signature may comprise a mutationat codons 31B, 40, 56, 57, 81 and/or 89, such as a mutation at each ofcodons 31B, 40, 56, 57, 81 and 89.

The method may further comprise assessing one or more traditional MSrisk factors. Assessing may comprise sequencing, and may comprises PCR.The B-cell is obtained from cerebrospinal fluid (CSF), and the methodmay further comprise assessing J chain usage, J chain length, and/orCDR3 length. The B-cell may be obtained from peripheral blood, and themethod may further comprise assessing J chain usage, J chain length,and/or CDR3 length. The method may also further comprise making atreatment decision based on the presence of said codon signature.

In another embodiment, there is provided a method of screening for anagent useful in treating multiple sclerosis (MS) comprising (a)providing an antibody produced by a VH4-expressing B-cell, said antibodycomprising mutations at three or more codons selected from the groupconsisting of 31B, 32, 40, 56, 57, 60, 81 and 89; (b) contacting saidantibody with a candidate ligand; and (c) assessing binding of saidcandidate ligand to said antibody, wherein binding of said candidateligand to said antibody identifies said candidate ligand as useful intreating MS.

In yet another embodiment, there is provided a method of treating asubject having or at risk of developing MS comprising administering tosaid subject a ligand that binds to an antibody VH-4 antibody comprisingmutations at three or more codons selected from the group consisting of31B, 32, 40, 56, 57, 60, 81 and 89. The ligand may a peptide or apeptoid, and may be linked to a toxin or B-cell antagonist.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

These, and other, embodiments of the invention will be betterappreciated and understood when considered in conjunction with thefollowing description and the accompanying drawings. It should beunderstood, however, that the following description, while indicatingvarious embodiments of the invention and numerous specific detailsthereof, is given by way of illustration and not of limitation. Manysubstitutions, modifications, additions and/or rearrangements may bemade within the scope of the invention without departing from the spiritthereof, and the invention includes all such substitutions,modifications, additions and/or rearrangements.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A-D. Frequency of VH Family Usage in Productive B CellRearrangements.

CD19+ B cells isolated from PB and CSF of (FIG. 1A) healthy controlperipheral blood (HCPB) (n=2) and multiple sclerosis patient peripheralblood (MSPB) (n=3), (FIG. 1B) multiple sclerosis cerebrospinal fluid(MSCSF), memory cells of MSCSF (mMSCSF), MSCSF normalized for clonalrepresentation, and MSCSF CD138+ plasma cells (all n=13), (FIG. 1C)clinically isolated syndrome cerebrospinal fluid (CISCSF) (n=3), memorycells of CISCSF (mCISCSF) (n=3), and MSCSF CD138 cells (n=1), and (FIG.1D) other neurological disease cerebrospinal fluid (ONDCSF) (n=2), cellsfrom the inflamed region of the parotid gland of a Sjögren's syndromepatient (n=1), systemic lupus erythematosus peripheral blood (SLEPB)(n=1). Data statistics can be found in Tables 9-12. The “n” numberdesignated in each panel is the number of productive VH sequences usedin each group; the “n” number in the legend is the number of patients ineach group.

FIG. 2. Frequency of J Segment Usage in Productive VH4 B CellRearrangements.

CD19+ B cells expressing a productive VH4 rearrangement were isolatedfrom HCPB, MSPB, and MSCSF and analyzed for J segment usage. The numberof patients included in each group can be found in the FIGS. 1A-Dlegend; the number of productive VH4 sequences in each group is shown inthe figure. Data statistics can be found in Table 13. The overallrepertoire J segment usages can be found in FIG. 6, with statistics inSupplemental Table 8.

FIGS. 3A-B. CDR3 Lengths of Productive VH Overall Repertoire, VH4Subdatabase, and VH3 Subdatabase.

(FIG. 3A) The average amino acid length of the CDR3 region. (FIG. 3B)Percentage of productive VH4 sequences in each range of CDR3 amino acidlengths. The number of patients included in each group can be found inthe FIGS. 1A-D legend; the number of VH4 productive sequences used foranalysis in each group is shown in the figure. Data statistics can befound in Table 15 for FIG. 3A and Table 16 for FIG. 3B.

FIG. 4. Ranges of Mutational Frequency in Overall Repertoire, VH4Subdatabase, and VH3 Subdatabase.

Mutations in CDR1, FR2, CDR2, and FR3 were included in the analysis.Since the average read length of VH sequences is 206 nucleotides, the0-2% range corresponds to 0-4 mutations, the 2-7% range corresponds to5-14 mutations, the 7-12% range corresponds to 15-24 mutations, and thegreater than 12% range corresponds to 25 or more mutations. The numberof patients included in each group can be found in the FIGS. 1A-Dlegend; the number of productive sequences used for analysis in eachgroup is shown in the figure. Data statistics can be found in Table 17.

FIGS. 5A-B. Model of VH4 Structure.

VH4-30.4 antibody structure was adapted from (Guddat et al., 1993) asdescribed in the Materials & Methods. The light chain variable domain isincluded for reference and is encoded in gray, while the heavy chainbackbone is in yellow. The VH4 signature has been demarcated, with “hot”spots in blue (residues 31B, 40, 56, 57, 60, 69, 81, and 89; correspondsto FIG. 7), and “cold” spots in green (residues 30, 52, and 68;corresponds to FIG. 7). Those hot or cold spots contained within a CDRhave been highlighted. The CDR3 is that of the original structure, andnot from any VH4 discussed here.

FIG. 6. Frequency of J Segment Usage in Overall Repertoire of ProductiveB Cell Rearrangements.

CD19+ B cells expressing a productive rearrangement were isolated fromHCPB, MSPB, and MSCSF and analyzed for J segment usage. The number ofpatients included in each group can be found in the FIGS. 1A-D legend;the number of productive sequences in each group is shown in the figure.Data statistics can be found in Table 15.

FIG. 7. Comparison of Sequence for VH4 Genes, Including a Consensus.

Individual genes are listed at the left hand side; “C” stand forconsensus.

FIG. 8. Example of VH4 Comparison. A VH4-30.4 Sequence is Listed as theGermline Configuration (Allele 01) and Compared to a Patient CD19+ BCell Sequence.

The germline protein conversion and the changes made by replacementmutations in the patient sequence are noted. Signature codons are boxed,with the dashed boxes demarcating cold spots, and the solid boxesdemarcating hot spots. CDRs as defined by Kabat (Kabat et al., 1983) areshaded.

FIG. 9. Signature Score in Individual MS and CIS Patients.

Signature scores were generated by calculating Z-scores for the RFvalues at the 6 codons within the signature (31B, 40, 56, 57, 81 and89). Individual Z-scores at each of the codon positions were compiled togenerate the composite signature Z-score. MS patient signature scoresare shown as black circles (), CIS patient signature scores thatresulted in prediction of CDMS are black squares (▪), and CIS patientsignature scores that resulted in prediction of unlikely to convert todefinite MS are in open squares (□). The average composite signaturescore in the MSCSFVH4 database was 10.9±2.0 (black line) and so anysignature score of an individual CIS patient above 6.8 (average S.D.;threshold shown as red line) was predicted to convert to CDMS. Forreference, ONDCSFVH4 group signature score was 4.5, and MSPBVH4signature score was 2.0.

FIGS. 10A-B. Model of VH4 Structure.

A VH4-30.4 antibody structure was adapted as described in the Materials& Methods. Two orientations of the structure are provided in FIGS. 10Aand 10B. The light chain variable domain is included for reference andis encoded in gray, while the heavy chain backbone is in yellow. The VH4signature has been demarcated, with “hot” spots in blue (residues 31B,32, 40, 56, 57, 60, 81, and 89), and “cold” spots in green (residues 30,43, 77, and 82). Residues contained within CDR1 and 2 are boxed. TheCDR3 is that of the original structure, and not from any VH4rearrangement discussed here.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Since 1998, a number of independent laboratories have documented thatVH4-expressing B cells are overrepresented in the CSF (Colombo et al.,2000; Monson et al., 2005; Owens et al., 2003; Qin et al., 1998; Ritchieet al., 2004) and brain lesions (Baranzini et al., 1999; Owens et al.,1998) of MS patients. This finding was initially inconspicuous sinceclonally expanding and autoreactive B cells from the CSF of MS patientscould be found utilizing variable genes from any of the heavy (andlight) chain families (Buluwela and Rabbitts, 1988; Humphries et al.,1988; Kodaira et al. 1986; Lee et al., 1987; Shen et al., 1987).However, emerging evidence that the VH4-expressing B cell populationharbors autoreactive B cells (Koelsch et al., 2007), combined with theestablished observation that VH4-expressing B cells are overrepresentedin CNS-derived B cell populations from MS patients (Colombo et al.,2000; Owens et al., 2003; Qin et al., 1998; Ritchie et al., 2004;Baranzini et al., 1999; Owens et al., 1998; Harp et al., 2007; Owens etal., 2007), prompted us to question the role of VH4-expressing B cellsin the CSF of MS patients.

To address this issue, the inventor compared repertoire characteristicsfrom their database of 405 CSF-derived B cells from 13 MS patients tothat of healthy controls as well as to several other B cell mediatedautoimmune diseases or other CNS-related disorders. The inventorpredicted that VH4-expressing B cells from the CSF of MS patients couldbe enriched for features associated with autoreactivity since (i) VH4expressing B cells from patients with autoimmune diseases (including SLEand RA) are enriched for autoreactivity (Pugh-Bernard et al., 2001;Zheng et al., 2004; Mockridge et al., 2004; Voswinkel et al., 1997;Hayashi et al., 2007; Huang et al., 1998), and (ii) some autoreactive,clonally-expanded CSF-derived B cells from MS patients use VH4 in theirantibody rearrangements (Lambracht-Washington et al., 2007). Featuresthat we were particularly interested in were those known to beassociated with autoreactivity including bias towards VH4-34 usage(Zheng et al., 2004) and features associated with receptor editingincluding bias in JH6 usage and long CDR3 lengths (Zheng et al., 2004;Meffre et al., 2000). Diminished mutational frequency has beenassociated with receptor editing (Meffre et al., 2000), and diminishedmutation targeting has been associated with clonally expanded CSFderived B cell populations in MS patients (Monson et al., 2005) and thuswere also included in the analyses.

In order to perform this VH4-specific analysis, the inventor constructedan extensive CSF B cell database containing 405 CSF-derived B cells from13 MS patients. Overrepresentation of VH4-expressing CD19+ B cells inthe CSF of MS patients was unique since VH4 overrepresentation was notobserved in B cell repertoires from the peripheral blood of (i) healthycontrol donors, (ii) patients with other autoimmune diseases with B cellinvolvement, including systemic lupus erythematosis (SLE) or Sjögren'ssyndrome, or (iii) MS patients from the same cohort. In fact, in depthanalysis of those VH4-expressing B cells from the peripheral blood of MSpatients within this cohort indicated that this group of B cells waslikely recognized for their autoreactive potential (as evidenced by highJH6 usage and long CDR3 length), and were denied further selection (asevidenced by low mutational frequencies). The inventor also did notobserve overrepresentation of VH4-expressing B cells in the CSF ofpatients with other neurological diseases, indicating thatover-representation of VH4 expressing B cells in the CSF of MS patientsis not due to bias in the ability of VH4 expressing B cells to enter theCNS. Taken together, these data suggest that VH4 expressing B cells areselected into the CSF B cell repertoire of MS patients in particular,and is further validated by the high mutational frequencies andpunctuated mutational targeting observed in this population.

Of the three CIS patients included in this comparison study, even thosethat convert to CDMS within the next year (CIS429 and CIS03-01) did nothave the overrepresentation of VH4 family usage in their CSF-derivedCD19+ B cell population. In contrast, evidence of VH4 overrepresentationis observed in the CD138+ plasma cells from CIS03-01. Since the plasmacells and plasma blasts are most likely arising from the CD19+ B cellpopulation (matching clones can be found in both compartments) (MartinMdel and Monson, 2007), it is reasonable to hypothesize that VH4expressing B cells which recognize their antigen in the CNS do notlinger in the memory pool long, but are signaled to differentiaterapidly into plasma blasts and plasma cells. This hypothesis is alsofurther substantiated by the lack of receptor editing in the VH4expressing CSF-derived B cells from these patients (as assessed bynormal JH6 usage and CDR3 length), as well as documentation thatplasmablasts and plasma cells are highly enriched in the CSF of thesepatients (Cepok et al., 2005; Winges et al., 2007). Dysregulation ofthese VH4 cells at the initiation of disease processes may be a centralcomponent of ongoing pathogenesis.

The inventor expected the increase in VH4 family usage would correspondto an increase in particular VH4 genes used most frequently in MSlesions and in the clones found in MSCSF such as 4-34, 4-39 and 4-59(Monson et al., 2005; Owens et al., 1998). However, usage frequency ofindividual VH4 genes within the VH4-expressing CSF B cell subdatabasewas no different than in PB of any cohort the inventor analyzed with theexception of VH4-34, which was utilized more frequently in SLE andSjögren's than in MSCSF. It is possible that B cells from the MSpatients examined were responding to a variety of VH4-binding antigens,so that the combination of these made an increase in a single geneindeterminable. Another possibility is an antigen may bind to the VH4genes and cause a superantigen response in only the B cells expressingVH4, similarly to what is seen with staphylococcal enterotoxin A withVH3-expressing B cells (Domiati-Saad and Lipsky, 1998). However,superantigen binding capacity is diminished with high mutationaccumulation (Oppezzo et al., 2004), and so a classical superantigenresponse is unlikely. In contrast, EBV infected memory B cells tend tohave high mutational frequencies and prevalent mutational targeting(Souza et al., 2007) similar to what we described in the MSCSF databasepresented here, but no mechanism of EBV infection susceptibility orimmune response to the virus has been reported that favorsVH4-expressing B cells over other heavy chain family expression.Nevertheless, the elevated mutational frequency observed inVH4-expressing B cells from the CSF of MS patients extends theinventor's previous hypothesis that CSF-derived B cells responding toantigen in the CNS are heavily driven within the CNS itself to suggestthat much of this heightened activity is occurring within theVH4-expressing CSF-derived B cell populations. Whether these B cells areresponding to self-antigens or valid foreign targets remainscontroversial. However, mutational analysis indicates that theVH4-expressing CSF-derived B cells from MS patients had gone through atypical germinal center, since mutational targeting to CDR and toDGYW/WRCH motifs is intact, unlike what has been observed in theindividual clonal populations from MS patients in the cohort (Monson etal., 2005). In addition, targeting was actually increased in theMSCSFVH4 subdatabase, most likely because the number of rounds ofsomatic hypermutation the B cells had undergone in response to antigenwas extensive (evidenced by the high mutation frequency). Defining theantigen specificity of highly mutated, VH4-expressing CSF-derived Bcells from MS patients will be paramount to resolving the mechanism ofthis unique selection of VH4 expressing B cells in the CSF of MSpatients.

Hyperintense mutation accumulation in the MSCSF database enabled theinventor to identify a unique 5 codon signature of VH4 replacementmutations—codons 31B, 40, 57, 60 and 69—that was not observed in thecontrol databases. Of these 5 codons, 31B was particularly interestingbecause it accumulated replacement mutations at a rate 7-fold higherthan expected, suggesting that this codon plays a pivotal role inantigen-antibody interactions. It is possible that myelin basic protein(MBP) may be excluded from this list of possible antigens interactingwith this unique antibody signature since one of the clonally expandedCSF-derived B cells strongly reactive to MBP (Lambracht-Washington etal., 2007) utilized a VH4-59 gene, which does not contain codon 31B.Also, since other databases in this analyses rarely (if ever)accumulated mutations in this position (0.17% in HCPB), it is likelythat the antigen targets of VH4 expressing CSF-derived B cells from MSpatients are not seen to a great extent in peripheral blood from healthydonors.

Codon composition can also influence the protein structure of antibodyvariable regions (Chothia et al., 1992). VH4-34 and 4-59 have a similarstructure, as they have neither codons 31A or 31B; VH4-04, 4-B, and 4-28have only codon 31A; and the 4-30 sub-genes, 4-39, 4-61, and 4-31 haveboth codons 31A and 31B. FIG. 7. In addition, several crucial codons areneeded to maintain structure; none of the VH4 signature codons are keyresidues that would change the structure of the antibody (Chothia etal., 1992; Chothia and Lesk, 1987). This infers that genes of similarstructure have similar antigen-binding sites, though the exact placementmay differ due to the size, hydrophilicity, and polarity of surroundingresidues. By the method designated by Clothia et al. (1992), CDR1 iscomprised of residues 26 through 32 because these are outside theframework β-sheets and form a loop involved in the antigen bindingpocket, and CDR2 is only residues 50 through 58; this translates intocodon 30, 31B, 52, 56, and 57 are all in direct contact with the antigen(FIG. 5), while 60 is between the antigen binding pocket and anothersurface loop not directly involved with antigen binding (Chothia et al.,1992). Therefore, codons 30 and 52 are likely “cold,” to maintainefficient antibody interaction with the antigen, while variation incodons 31B (in the few genes it is in), 56, and 57 provide moreeffective binding to their antigen with different size, hydrophilicity,or polarity properties. It is less clear why residues 40, 69, 81, and 89are “hot” or residue 68 is “cold,” and how replacement mutations atthese positions affect VH4 antigen binding (FIG. 5). Investigating theimpact of replacement mutations at these positions will provideimportant clues regarding the interaction of these VH4 utilizingantibodies with self-antigens in the CNS.

It is also likely that different combinations of residue replacementsaffect binding to discreet antigens. For example, perhaps thecombination of replacements at codons A, B and C mediate high affinitybinding to antigen X, while replacements at codons BDE mediate highaffinity binding to antigen Y. This would explain the differences inreplacement mutation positions in different VH4 genes; codon positionsABC are needed for 4-31 to bind antigen X, while codon positions BDE areneeded for 4-39 to bind antigen Y. In support of this, the inventorfound that different VH4 genes do selectively use the MS signaturemutations at varying levels; for example, VH4-30 has more mutations incodons 56 and 81, while VH4-39 tends to accumulate mutations morerapidly in codons 31B, 50, 56, and 81 (Table 6).

In summary, VH4 family usage is substantially increased in both CD19+ Bcells and CD138+ plasma cells isolated from the central nervous systemof MS patients, (FIG. 1 and (Owens et al., 2007)), but as shown here,not in healthy controls, patients with other CNS-related diseases, orpatients with other B cell related autoimmune diseases. The VH4overexpression seen in the MS patients is due to changes in use of manyof the genes in the VH4 family (rather than VH4-34 alone), andmutational analysis suggests that antigen-driven selection in thecontext of classical germinal centers is preserved. Thus, the VH4expressing B cells from the CSF of MS patients are not dysregulated atthis level of selection. More importantly, a unique 11 codon footprintof mutational characteristics can be found in the MSCSF VH4 subdatabasethat is not observed in healthy control peripheral blood or CSF-derivedB cells from patients with other neurological diseases. This signature,which accumulates replacement mutations up to 7-fold more frequentlythan in healthy control PB-derived B cells, is most likely a combinationof sub-signatures that mediate effective binding to antigens present inthe CNS. The inventor now proposes the use of this signature to predictor diagnose MS in subjects.

1. VH4

The normal immune system has the ability to generate millions ofantibodies with different antigen binding abilities. The diversity isbrought about by the complexities of constructing immunoglobulinmolecules. These molecules consist of paired polypeptide chains (heavyand light) each containing a constant and a variable region. Thestructures of the variable regions of the heavy and light chains arespecified by immunoglobulin V genes. The heavy chain variable region isderived from three gene segments known as VH, D and JH. In humans thereare about 100 different VH segments, over 20 D segments and six JHsegments. The light chain genes have only two segments, the VL and JLsegments. Antibody diversity is the result of random combinations ofVH/D/JH segments with VUJL components superimposed on which are severalmechanisms including junctional diversity and somatic mutation.

The germline VH genes can be separated into at least six families (VH1through VH6) based on DNA nucleotide sequence identity of the first 95to 101 amino acids. Members of the same family typically have ≧80%sequence identity, whereas members of different families have less than70% identity. These families range in size from one VH6 gene to anestimated greater than 45 VH3 genes. In addition, many pseudogenesexist. Recent studies have nearly completed a physical map of the VHlocus on chromosome 14q32.13.15. It has now been estimated that thehuman VH repertoire is represented by approximately 50 functional VHsegments with about an equal number of pseudogenes. These studiesestimate the size of the VH locus to be approximately 1100 kb, which isless than half the previous estimates of 2.5 to 3 megabases asdetermined by pulse field gel electrophoreis. The VH4 family of genescontains 9 different members: 4-04, 4-28, 4-30, 4-31, 4-34, 4-39, 4-59,4-61, 4-B4 (see FIG. 7).

The present invention relates to identifcation of a “signature” in theVH4 sequences of certain B cells. The sequence signature initiallycomprises residues 31B, 56 and/or 81, but also can include one or moreof residues 30, 40, 52, 57, 60, 68, 69 and 89 (FIG. 7). By examining thesequence at these positions, and identifying mutations at one or more ofthe positions, it can be determined that a subject is at risk ofdeveloping MS and, in the presence of additional factors, has MS.

II. NUCLEIC ACIDS AND DETECTION METHODS THEREFOR

Another aspect of the present invention concerns isolated DNA segmentsand their use in detecting the presence of mutations in certain codonsof the VH4 segments from a subject. Many methods described herein willinvolve the use of amplification primers, oligonucleotide probes, andother nucleic acid elements involved in the analysis of genomic DNA,cDNA or mRNA transcripts, such as SEQ ID NO:2, which is the germline ornormal sequence of VH4 family genes.

The term “nucleic acid” is well known in the art. A “nucleic acid” asused herein will generally refer to a molecule (i.e., a strand) of DNAor RNA comprising a nucleobase. A nucleobase includes, for example, anaturally-occurring purine or pyrimidine base found in DNA (e.g., anadenine “A,” a guanine “G,” a thymine “T” or a cytosine “C”) or RNA(e.g., an A, a G, an uracil “U” or a C). The term “nucleic acid”encompass the terms “oligonucleotide” and “polynucleotide,” each as asubgenus of the term “nucleic acid.” The term “oligonucleotide” refersto a molecule of between about 3 and about 100 nucleobases in length.The term “polynucleotide” refers to at least one molecule of greaterthan about 100 nucleobases in length. A “gene” refers to coding sequenceof a gene product, as well as introns and the promoter of the geneproduct.

These definitions generally refer to a single-stranded molecule, but inspecific embodiments will also encompass an additional strand that ispartially, substantially or fully complementary to the single-strandedmolecule. Thus, a nucleic acid may encompass a double-stranded moleculethat comprises complementary strands or “complements” of a particularsequence comprising a molecule. In particular aspects, a nucleic acidencodes a protein or polypeptide, or a portion thereof.

A. Preparation of Nucleic Acids

A nucleic acid may be made by any technique known to one of ordinaryskill in the art, such as for example, chemical synthesis, enzymaticproduction or biological production. Non-limiting examples of asynthetic nucleic acid (e.g., a synthetic oligonucleotide), include anucleic acid made by in vitro chemical synthesis using phosphotriester,phosphite or phosphoramidite chemistry and solid phase techniques suchas described in EP 266,032, incorporated herein by reference, or viadeoxynucleoside H-phosphonate intermediates as described by Froehler etal., 1986 and U.S. Pat. No. 5,705,629, each incorporated herein byreference. In the methods of the present invention, one or moreoligonucleotide may be used. Various different mechanisms ofoligonucleotide synthesis have been disclosed in for example, U.S. Pat.Nos. 4,659,774, 4,816,571, 5,141,813, 5,264,566, 4,959,463, 5,428,148,5,554,744, 5,574,146, 5,602,244, each of which is incorporated herein byreference.

A non-limiting example of an enzymatically produced nucleic acid includeone produced by enzymes in amplification reactions such as PCR™ (see forexample, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,682,195, eachincorporated herein by reference), or the synthesis of anoligonucleotide described in U.S. Pat. No. 5,645,897, incorporatedherein by reference. A non-limiting example of a biologically producednucleic acid includes a recombinant nucleic acid produced (i.e.,replicated) in a living cell, such as a recombinant DNA vectorreplicated in bacteria (see for example, Sambrook et al. 2001,incorporated herein by reference).

B. Purification of Nucleic Acids

A nucleic acid may be purified on polyacrylamide gels, cesium chloridecentrifugation gradients, chromatography columns or by any other meansknown to one of ordinary skill in the art (see for example, Sambrook etal., 2001, incorporated herein by reference). In some aspects, a nucleicacid is a pharmacologically acceptable nucleic acid. Pharmacologicallyacceptable compositions are known to those of skill in the art, and aredescribed herein.

In certain aspects, the present invention concerns a nucleic acid thatis an isolated nucleic acid. As used herein, the term “isolated nucleicacid” refers to a nucleic acid molecule (e.g., an RNA or DNA molecule)that has been isolated free of, or is otherwise free of, the bulk of thetotal genomic and transcribed nucleic acids of one or more cells. Incertain embodiments, “isolated nucleic acid” refers to a nucleic acidthat has been isolated free of, or is otherwise free of, bulk ofcellular components or in vitro reaction components such as for example,macromolecules such as lipids or proteins, small biological molecules,and the like.

C. Nucleic Acid Complements

As discussed above, the present invention encompasses a nucleic acidthat is complementary to a nucleic acid. A nucleic acid is “complements”or is “complementary” to another nucleic acid when it is capable ofbase-pairing with another nucleic acid according to the standardWatson-Crick, Hoogsteen or reverse Hoogsteen binding complementarityrules. As used herein “another nucleic acid” may refer to a separatemolecule or a spatial separated sequence of the same molecule. Inpreferred embodiments, a complement is a hybridization probe oramplification primer for the detection of a nucleic acid polymorphism.

As used herein, the term “complementary” or “complement” also refers toa nucleic acid comprising a sequence of consecutive nucleobases orsemiconsecutive nucleobases (e.g., one or more nucleobase moieties arenot present in the molecule) capable of hybridizing to another nucleicacid strand or duplex even if less than all the nucleobases do not basepair with a counterpart nucleobase. However, in some diagnostic ordetection embodiments, completely complementary nucleic acids arepreferred.

D. Nucleic Acid Detection and Evaluation

Those in the art will readily recognize that nucleic acid molecules maybe double-stranded molecules and that reference to a particular site onone strand refers, as well, to the corresponding site on a complementarystrand. Thus, in defining a polymorphic site, reference to an adenine, athymine (uridine), a cytosine, or a guanine at a particular site on theplus (sense or coding) strand of a nucleic acid molecule is alsointended to include the thymine (uridine), adenine, guanine, or cytosine(respectively) at the corresponding site on a minus (antisense ornoncoding) strand of a complementary strand of a nucleic acid molecule.Thus, reference may be made to either strand and still comprise the samepolymorphic site and an oligonucleotide may be designed to hybridize toeither strand. Throughout the text, in identifying a polymorphic site,reference is made to the sense strand, only for the purpose ofconvenience.

Typically, the nucleic acid mixture is isolated from a biological sampletaken from the individual, such as a blood, fecal or tissue (e.g.,intestinal mucosal) sample using standard techniques such as disclosedin Jones (1963) which is hereby incorporated by reference. Othersuitable tissue samples include whole blood, saliva, tears, urine,sweat, buccal, skin and hair. The nucleic acid mixture may be comprisedof genomic DNA, mRNA, or cDNA. Furthermore it will be understood by theskilled artisan that mRNA or cDNA preparations would not be used todetect polymorphisms located in introns or in 5′ and 3′ non-transcribedregions.

The identity of a nucleotide (or nucleotide pair) at a polymorphic sitemay be determined by amplifying a target region(s) containing thepolymorphic site(s) directly from one or both copies of the gene presentin the individual and the sequence of the amplified region(s) determinedby conventional methods. It will be readily appreciated by the skilledartisan that only one nucleotide will be detected at a polymorphic sitein individuals who are homozygous at that site, while two differentnucleotides will be detected if the individual is heterozygous for thatsite. The polymorphism may be identified directly, known aspositive-type identification, or by inference, referred to asnegative-type identification. For example, where a SNP is known to beguanine and cytosine in a reference population, a site may be positivelydetermined to be either guanine or cytosine for an individual homozygousat that site, or both guanine and cytosine, if the individual isheterozygous at that site. Alternatively, the site may be negativelydetermined to be not guanine (and thus cytosine/cytosine) or notcytosine (and thus guanine/guanine).

The target region(s) may be amplified using any oligonucleotide-directedamplification method, including but not limited to polymerase chainreaction (PCR) (U.S. Pat. No. 4,965,188), ligase chain reaction (LCR)(Barany et al., 1991; WO90/01069), and oligonucleotide ligation assay(OLA) (Landegren et al., 1988). Oligonucleotides useful as primers orprobes in such methods should specifically hybridize to a region of thenucleic acid that contains or is adjacent to the polymorphic site.Typically, the oligonucleotides are between 10 and 35 nucleotides inlength and preferably, between 15 and 30 nucleotides in length. Mostpreferably, the oligonucleotides are 20 to 25 nucleotides long. Theexact length of the oligonucleotide will depend on many factors that areroutinely considered and practiced by the skilled artisan.

Other known nucleic acid amplification procedures may be used to amplifythe target region including transcription-based amplification systems(U.S. Pat. No. 5,130,238; EP 329,822; U.S. Pat. No. 5,169,766,WO89/06700) and isothermal methods (Walker et al., 1992).

A polymorphism in the target region may also be assayed before or afteramplification using one of several hybridization-based methods known inthe art. Typically, allele-specific oligonucleotides are utilized inperforming such methods. The allele-specific oligonucleotides may beused as differently labeled probe pairs, with one member of the pairshowing a perfect match to one variant of a target sequence and theother member showing a perfect match to a different variant. In someembodiments, more than one polymorphic site may be detected at onceusing a set of allele-specific oligonucleotides or oligonucleotidepairs.

Hybridization of an allele-specific oligonucleotide to a targetpolynucleotide may be performed with both entities in solution, or suchhybridization may be performed when either the oligonucleotide or thetarget polynucleotide is covalently or noncovalently affixed to a solidsupport. Attachment may be mediated, for example, by antibody-antigeninteractions, poly-L-Lys, streptavidin or avidin-biotin, salt bridges,hydrophobic interactions, chemical linkages, UV cross-linking baking,etc. Allele-specific oligonucleotides may be synthesized directly on thesolid support or attached to the solid support subsequent to synthesis.Solid-supports suitable for use in detection methods of the inventioninclude substrates made of silicon, glass, plastic, paper and the like,which may be formed, for example, into wells (as in 96-well plates),slides, sheets, membranes, fibers, chips, dishes, and beads. The solidsupport may be treated, coated or derivatized to facilitate theimmobilization of the allele-specific oligonucleotide or target nucleicacid.

The genotype for one or more polymorphic sites in the gene of anindividual may also be determined by hybridization of one or both copiesof the gene, or a fragment thereof, to nucleic acid arrays and subarrayssuch as described in WO 95/11995. The arrays would contain a battery ofallele-specific oligonucleotides representing each of the polymorphicsites to be included in the genotype or haplotype.

The identity of polymorphisms may also be determined using a mismatchdetection technique, including but not limited to the RNase protectionmethod using riboprobes (Winter et al., 1985; Meyers et al., 1985) andproteins which recognize nucleotide mismatches, such as the E. coli mutSprotein (Modrich, 1991). Alternatively, variant alleles can beidentified by single strand conformation polymorphism (SSCP) analysis(Orita et al., 1989; Humphries, et al., 1996) or denaturing gradient gelelectrophoresis (DGGE) (Wartell et al., 1990; Sheffield et al., 1989).

A polymerase-mediated primer extension method may also be used toidentify the polymorphism(s). Several such methods have been describedin the patent and scientific literature. Extended primers containing apolymorphism may be detected by mass spectrometry as described in U.S.Pat. No. 5,605,798. Another primer extension method is allele-specificPCR (Ruano et al., 1989; Ruano et al., 1991; WO 93/22456; Turki et al.,1995).

1. Hybridization

The use of a probe or primer of between 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 60, 70, 80, 90, or 100nucleotides, preferably between 17 and 100 nucleotides in length, or insome aspects of the invention up to 1-2 kilobases or more in length,allows the formation of a duplex molecule that is both stable andselective. Molecules having complementary sequences over contiguousstretches greater than 20 bases in length are generally preferred, toincrease stability and/or selectivity of the hybrid molecules obtained.One will generally prefer to design nucleic acid molecules forhybridization having one or more complementary sequences of 20 to 30nucleotides, or even longer where desired. Such fragments may be readilyprepared, for example, by directly synthesizing the fragment by chemicalmeans or by introducing selected sequences into recombinant vectors forrecombinant production.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of DNAs and/or RNAs or to provide primers for amplification ofDNA or RNA from samples. Depending on the application envisioned, onewould desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of the probe or primers for the targetsequence.

For applications requiring high selectivity, one will typically desireto employ relatively high stringency conditions to form the hybrids. Forexample, relatively low salt and/or high temperature conditions, such asprovided by about 0.02 M to about 0.10 M NaCl at temperatures of about50° C. to about 70° C. Such high stringency conditions tolerate little,if any, mismatch between the probe or primers and the template or targetstrand and would be particularly suitable for isolating specific genesor for detecting a specific polymorphism. It is generally appreciatedthat conditions can be rendered more stringent by the addition ofincreasing amounts of formamide. For example, under highly stringentconditions, hybridization to filter-bound DNA may be carried out in 0.5M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., andwashing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel et al., 1989).

Conditions may be rendered less stringent by increasing saltconcentration and/or decreasing temperature. For example, a mediumstringency condition could be provided by about 0.1 to 0.25 M NaCl attemperatures of about 37° C. to about 55° C., while a low stringencycondition could be provided by about 0.15 M to about 0.9 M salt, attemperatures ranging from about 20° C. to about 55° C. Under lowstringent conditions, such as moderately stringent conditions thewashing may be carried out for example in 0.2×SSC/0.1% SDS at 42° C.(Ausubel et al., 1989). Hybridization conditions can be readilymanipulated depending on the desired results.

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 1.0 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C.

In certain embodiments, it will be advantageous to employ nucleic acidsof defined sequences of the present invention in combination with anappropriate means, such as a label, for determining hybridization. Awide variety of appropriate indicator means are known in the art,including fluorescent, radioactive, enzymatic or other ligands, such asavidin/biotin, which are capable of being detected. In preferredembodiments, one may desire to employ a fluorescent label or an enzymetag such as urease, alkaline phosphatase or peroxidase, instead ofradioactive or other environmentally undesirable reagents. In the caseof enzyme tags, colorimetric indicator substrates are known that can beemployed to provide a detection means that is visibly orspectrophotometrically detectable, to identify specific hybridizationwith complementary nucleic acid containing samples. In other aspects, aparticular nuclease cleavage site may be present and detection of aparticular nucleotide sequence can be determined by the presence orabsence of nucleic acid cleavage.

In general, it is envisioned that the probes or primers described hereinwill be useful as reagents in solution hybridization, as in PCR, fordetection of expression or genotype of corresponding genes, as well asin embodiments employing a solid phase. In embodiments involving a solidphase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to hybridization with selected probes under desiredconditions. The conditions selected will depend on the particularcircumstances (depending, for example, on the G+C content, type oftarget nucleic acid, source of nucleic acid, size of hybridizationprobe, etc.). Optimization of hybridization conditions for theparticular application of interest is well known to those of skill inthe art. After washing of the hybridized molecules to removenon-specifically bound probe molecules, hybridization is detected,and/or quantified, by determining the amount of bound label.Representative solid phase hybridization methods are disclosed in U.S.Pat. Nos. 5,843,663, 5,900,481 and 5,919,626. Other methods ofhybridization that may be used in the practice of the present inventionare disclosed in U.S. Pat. Nos. 5,849,481, 5,849,486 and 5,851,772. Therelevant portions of these and other references identified in thissection of the Specification are incorporated herein by reference.

2. Amplification of Nucleic Acids

Nucleic acids used as a template for amplification may be isolated fromcells, tissues or other samples according to standard methodologies(Sambrook et al., 2001). In certain embodiments, analysis is performedon whole cell or tissue homogenates or biological fluid samples with orwithout substantial purification of the template nucleic acid. Thenucleic acid may be genomic DNA or fractionated or whole cell RNA. WhereRNA is used, it may be desired to first convert the RNA to acomplementary DNA.

The term “primer,” as used herein, is meant to encompass any nucleicacid that is capable of priming the synthesis of a nascent nucleic acidin a template-dependent process. Typically, primers are oligonucleotidesfrom ten to twenty and/or thirty base pairs in length, but longersequences can be employed. Primers may be provided in double-strandedand/or single-stranded form, although the single-stranded form ispreferred.

Pairs of primers designed to selectively hybridize to nucleic acidscorresponding to the variable heavy chain gene locus, variants andfragments thereof are contacted with the template nucleic acid underconditions that permit selective hybridization. Depending upon thedesired application, high stringency hybridization conditions may beselected that will only allow hybridization to sequences that arecompletely complementary to the primers. In other embodiments,hybridization may occur under reduced stringency to allow foramplification of nucleic acids that contain one or more mismatches withthe primer sequences. Once hybridized, the template-primer complex iscontacted with one or more enzymes that facilitate template-dependentnucleic acid synthesis. Multiple rounds of amplification, also referredto as “cycles,” are conducted until a sufficient amount of amplificationproduct is produced.

The amplification product may be detected, analyzed or quantified. Incertain applications, the detection may be performed by visual means. Incertain applications, the detection may involve indirect identificationof the product via chemiluminescence, radioactive scintigraphy ofincorporated radiolabel or fluorescent label or even via a system usingelectrical and/or thermal impulse signals (Affymax technology; Bellus,1994).

A number of template dependent processes are available to amplify theoligonucleotide sequences present in a given template sample. One of thebest known amplification methods is the polymerase chain reaction(referred to as PCR™) which is described in detail in U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159, and in Innis et al., 1988, each ofwhich is incorporated herein by reference in their entirety.

Primer extension, which may be used as a stand alone technique or incombination with other methods (such as PCR), requires a labeled primer(usually 20-50 nucleotides in length) which is complementary to a regionnear the 5′ end of the gene. The primer is allowed to anneal to the RNAand reverse transcriptase is used to synthesize complementary cDNA tothe RNA until it reaches the 5′ end of the RNA.

Another method for amplification is ligase chain reaction (“LCR”),disclosed in European Application No. 320 308, incorporated herein byreference in its entirety. U.S. Pat. No. 4,883,750 describes a methodsimilar to LCR for binding probe pairs to a target sequence. A methodbased on PCR™ and oligonucleotide ligase assay (OLA) (described infurther detail below), disclosed in U.S. Pat. No. 5,912,148, may also beused.

Alternative methods for amplification of target nucleic acid sequencesthat may be used in the practice of the present invention are disclosedin U.S. Pat. Nos. 5,843,650, 5,846,709, 5,846,783, 5,849,546, 5,849,497,5,849,547, 5,858,652, 5,866,366, 5,916,776, 5,922,574, 5,928,905,5,928,906, 5,932,451, 5,935,825, 5,939,291 and 5,942,391, Great BritainApplication 2 202 328, and in PCT Application PCT/US89/01025, each ofwhich is incorporated herein by reference in its entirety. QbetaReplicase, described in PCT Application PCT/US87/00880, may also be usedas an amplification method in the present invention.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[alpha-thio]-triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids in the present invention (Walker et al., 1992). StrandDisplacement Amplification (SDA), disclosed in U.S. Pat. No. 5,916,779,is another method of carrying out isothermal amplification of nucleicacids which involves multiple rounds of strand displacement andsynthesis, i.e., nick translation

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR (Kwoh et al., 1989; PCT Application WO88/10315, incorporated herein by reference in their entirety). EuropeanApplication 329 822 disclose a nucleic acid amplification processinvolving cyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA,and double-stranded DNA (dsDNA), which may be used in accordance withthe present invention.

PCT Application WO 89/06700 (incorporated herein by reference in itsentirety) disclose a nucleic acid sequence amplification scheme based onthe hybridization of a promoter region/primer sequence to a targetsingle-stranded DNA (“ssDNA”) followed by transcription of many RNAcopies of the sequence. This scheme is not cyclic, i.e., new templatesare not produced from the resultant RNA transcripts. Other amplificationmethods include “RACE” and “one-sided PCR” (Frohman, 1990; Ohara et al.,1989).

Real-time polymerase chain reaction, also called quantitative real timepolymerase chain reaction (qPCR) or kinetic polymerase chain reaction,is a laboratory technique based on the polymerase chain reaction, whichis used to amplify and simultaneously quantify a targeted DNA molecule.It enables both detection and quantification (as absolute number ofcopies or relative amount when normalized to DNA input or additionalnormalizing genes) of a specific sequence in a DNA sample.

The procedure follows the general principle of polymerase chainreaction; its key feature is that the amplified DNA is quantified as itaccumulates in the reaction in real time after each amplification cycle.Two common methods of quantification are the use of fluorescent dyesthat intercalate with double-stranded DNA, and modified DNAoligonucleotide probes that fluoresce when hybridized with acomplementary DNA.

Frequently, real-time polymerase chain reaction is combined with reversetranscription polymerase chain reaction to quantify low abundancemessenger RNA (mRNA), enabling a researcher to quantify relative geneexpression at a particular time, or in a particular cell or tissue type.Although real-time quantitative polymerase chain reaction is oftenmarketed as RT-PCR, it should not be confused with reverse transcriptionpolymerase chain reaction, also known as RT-PCR.

A DNA-binding dye binds to all double-stranded (ds)DNA in a PCRreaction, causing fluorescence of the dye. An increase in DNA productduring PCR therefore leads to an increase in fluorescence intensity andis measured at each cycle, thus allowing DNA concentrations to bequantified. However, dsDNA dyes such as SYBR Green will bind to alldsDNA PCR products, including non-specific PCR products (such as “primerdimers”). This can potentially interfere with or prevent accuratequantification of the intended target sequence. The reaction is preparedas usual, with the addition of fluorescent dsDNA dye.

The reaction is run in a thermocycler, and after each cycle, the levelsof fluorescence are measured with a detector; the dye only fluoresceswhen bound to the dsDNA (i.e., the PCR product). With reference to astandard dilution, the dsDNA concentration in the PCR can be determined.

Like other real-time PCR methods, the values obtained do not haveabsolute units associated with it (i.e. mRNA copies/cell). As describedabove, a comparison of a measured DNA/RNA sample to a standard dilutionwill only give a fraction or ratio of the sample relative to thestandard, allowing only relative comparisons between different tissuesor experimental conditions. To ensure accuracy in the quantification, itis usually necessary to normalize expression of a target gene to astably expressed gene. This can correct possible differences in RNAquantity or quality across experimental samples.

Using fluorescent reporter probes is the most accurate and most reliableof the methods, but also the most expensive. It uses a sequence-specificRNA or DNA-based probe to quantify only the DNA containing the probesequence; therefore, use of the reporter probe significantly increasesspecificity, and allows quantification even in the presence of somenon-specific DNA amplification. This potentially allows formultiplexing—assaying for several genes in the same reaction by usingspecific probes with different-coloured labels, provided that all genesare amplified with similar efficiency.

It is commonly carried out with an RNA-based probe with a fluorescentreporter at one end and a quencher of fluorescence at the opposite endof the probe. The close proximity of the reporter to the quencherprevents detection of its fluorescence; breakdown of the probe by the 5′to 3′ exonuclease activity of the taq polymerase breaks thereporter-quencher proximity and thus allows unquenched emission offluorescence, which can be detected. An increase in the product targetedby the reporter probe at each PCR cycle therefore causes a proportionalincrease in fluorescence due to the breakdown of the probe and releaseof the reporter.

The PCR reaction is prepared as usual (see PCR), and the reporter probeis added. As the reaction commences, during the annealing stage of thePCR both probe and primers anneal to the DNA target. Polymerisation of anew DNA strand is initiated from the primers, and once the polymerasereaches the probe, its 5′-3-exonuclease degrades the probe, physicallyseparating the fluorescent reporter from the quencher, resulting in anincrease in fluorescence.

Fluorescence is detected and measured in the real-time PCR thermocycler,and its geometric increase corresponding to exponential increase of theproduct is used to determine the threshold cycle (C_(T)) in eachreaction.

Quantitating gene expression by traditional methods presents severalproblems. Firstly, detection of mRNA on a Northern blot or PCR productson a gel or Southern blot is time-consuming and does not allow precisequantitation. Also, over the 20-40 cycles of a typical PCR reaction, theamount of product reaches a plateau determined more by the amount ofprimers in the reaction mix than by the input template/sample.

Relative concentrations of DNA present during the exponential phase ofthe reaction are determined by plotting fluorescence against cyclenumber on a logarithmic scale (so an exponentially increasing quantitywill give a straight line). A threshold for detection of fluorescenceabove background is determined. The cycle at which the fluorescence froma sample crosses the threshold is called the cycle threshold, C_(r).Since the quantity of DNA doubles every cycle during the exponentialphase, relative amounts of DNA can be calculated, e.g., a sample whoseC_(t) is 3 cycles earlier than another's has 2³=8 times more template.

Amounts of RNA or DNA are then determined by comparing the results to astandard curve produced by RT-PCR of serial dilutions (e.g., undiluted,1:4, 1:16, 1:64) of a known amount of RNA or DNA. As mentioned above, toaccurately quantify gene expression, the measured amount of RNA from thegene of interest is divided by the amount of RNA from a housekeepinggene measured in the same sample to normalize for possible variation inthe amount and quality of RNA between different samples. Thisnormalization permits accurate comparison of expression of the gene ofinterest between different samples, provided that the expression of thereference (housekeeping) gene used in the normalization is very similaracross all the samples. Choosing a reference gene fulfilling thiscriterion is therefore of high importance, and often challenging,because only very few genes show equal levels of expression across arange of different conditions or tissues.

3. Detection of Nucleic Acids

Following any amplification, it may be desirable to separate theamplification product from the template and/or the excess primer. In oneembodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods (Sambrook et al., 2001). Separated amplification products may becut out and eluted from the gel for further manipulation. Using lowmelting point agarose gels, the separated band may be removed by heatingthe gel, followed by extraction of the nucleic acid.

Separation of nucleic acids may also be effected by spin columns and/orchromatographic techniques known in art. There are many kinds ofchromatography which may be used in the practice of the presentinvention, including adsorption, partition, ion-exchange,hydroxylapatite, molecular sieve, reverse-phase, column, paper,thin-layer, and gas chromatography as well as HPLC.

In certain embodiments, the amplification products are visualized, withor without separation. A typical visualization method involves stainingof a gel with ethidium bromide and visualization of bands under UVlight. Alternatively, if the amplification products are integrallylabeled with radio- or fluorometrically-labeled nucleotides, theseparated amplification products can be exposed to x-ray film orvisualized under the appropriate excitatory spectra.

In one embodiment, following separation of amplification products, alabeled nucleic acid probe is brought into contact with the amplifiedmarker sequence. The probe preferably is conjugated to a chromophore butmay be radiolabeled. In another embodiment, the probe is conjugated to abinding partner, such as an antibody or biotin, or another bindingpartner carrying a detectable moiety.

In particular embodiments, detection is by Southern blotting andhybridization with a labeled probe. The techniques involved in Southernblotting are well known to those of skill in the art (see Sambrook etal., 2001). One example of the foregoing is described in U.S. Pat. No.5,279,721, incorporated by reference herein, which discloses anapparatus and method for the automated electrophoresis and transfer ofnucleic acids. The apparatus permits electrophoresis and blottingwithout external manipulation of the gel and is ideally suited tocarrying out methods according to the present invention.

Other methods of nucleic acid detection that may be used in the practiceof the instant invention are disclosed in U.S. Pat. Nos. 5,840,873,5,843,640, 5,843,651, 5,846,708, 5,846,717, 5,846,726, 5,846,729,5,849,487, 5,853,990, 5,853,992, 5,853,993, 5,856,092, 5,861,244,5,863,732, 5,863,753, 5,866,331, 5,905,024, 5,910,407, 5,912,124,5,912,145, 5,919,630, 5,925,517, 5,928,862, 5,928,869, 5,929,227,5,932,413 and 5,935,791, each of which is incorporated herein byreference.

4. Other Assays

Other methods for genetic screening may be used within the scope of thepresent invention, for example, to detect mutations in genomic DNA, cDNAand/or RNA samples. Methods used to detect point mutations includedenaturing gradient gel electrophoresis (DGGE), restriction fragmentlength polymorphism analysis (RFLP), chemical or enzymatic cleavagemethods, direct sequencing of target regions amplified by PCR™ (seeabove), single-strand conformation polymorphism analysis (SSCP) andother methods well known in the art.

One method of screening for point mutations is based on RNase cleavageof base pair mismatches in RNA/DNA or RNA/RNA heteroduplexes. As usedherein, the term “mismatch” is defined as a region of one or moreunpaired or mispaired nucleotides in a double-stranded RNA/RNA, RNA/DNAor DNA/DNA molecule. This definition thus includes mismatches due toinsertion/deletion mutations, as well as single or multiple base pointmutations.

U.S. Pat. No. 4,946,773 describes an RNase A mismatch cleavage assaythat involves annealing single-stranded DNA or RNA test samples to anRNA probe, and subsequent treatment of the nucleic acid duplexes withRNase A. For the detection of mismatches, the single-stranded productsof the RNase A treatment, electrophoretically separated according tosize, are compared to similarly treated control duplexes. Samplescontaining smaller fragments (cleavage products) not seen in the controlduplex are scored as positive.

Other investigators have described the use of RNase I in mismatchassays. The use of RNase I for mismatch detection is described inliterature from Promega Biotech. Promega markets a kit containing RNaseI that is reported to cleave three out of four known mismatches. Othershave described using the MutS protein or other DNA-repair enzymes fordetection of single-base mismatches.

Alternative methods for detection of deletion, insertion or substitutionmutations that may be used in the practice of the present invention aredisclosed in U.S. Pat. Nos. 5,849,483, 5,851,770, 5,866,337, 5,925,525and 5,928,870, each of which is incorporated herein by reference in itsentirety.

5. Polymorphic Nucleic Acid Screening Methods

Spontaneous mutations that arise during the course of evolution in thegenomes of organisms are often not immediately transmitted throughoutall of the members of the species, thereby creating polymorphic allelesthat co-exist in the species populations. Often polymorphisms are thecause of genetic diseases. Several classes of polymorphisms have beenidentified. For example, variable nucleotide type polymorphisms (VNTRs),arise from spontaneous tandem duplications of di- or trinucleotiderepeated motifs of nucleotides. If such variations alter the lengths ofDNA fragments generated by restriction endonuclease cleavage, thevariations are referred to as restriction fragment length polymorphisms(RFLPs). RFLPs are been widely used in human and animal geneticanalyses.

Another class of polymorphisms is generated by the replacement of asingle nucleotide. Such single nucleotide polymorphisms (SNPs) rarelyresult in changes in a restriction endonuclease site. Thus, SNPs arerarely detectable restriction fragment length analysis. SNPs are themost common genetic variations and occur once every 100 to 300 bases andseveral SNP mutations have been found that affect a single nucleotide ina protein-encoding gene in a manner sufficient to actually cause agenetic disease. SNP diseases are exemplified by hemophilia, sickle-cellanemia, hereditary hemochromatosis, late-onset Alzheimer's disease, etc.

Several methods have been developed to screen polymorphisms and someexamples are listed below. The reference of Kwok and Chen (2003) andKwok (2001) provide overviews of some of these methods; both of thesereferences are specifically incorporated by reference. SNPs can becharacterized by the use of any of these methods or suitablemodification thereof. Such methods include the direct or indirectsequencing of the site, the use of restriction enzymes where therespective alleles of the site create or destroy a restriction site, theuse of allele-specific hybridization probes, the use of antibodies thatare specific for the proteins encoded by the different alleles of thepolymorphism, or any other biochemical interpretation.

i. DNA Sequencing

The most commonly used method of characterizing a polymorphism is directDNA sequencing of the genetic locus that flanks and includes thepolymorphism. Such analysis can be accomplished using either the“dideoxy-mediated chain termination method,” also known as the “SangerMethod” (Sanger et al., 1975) or the “chemical degradation method,” alsoknown as the “Maxam-Gilbert method” (Maxam et al., 1977). Sequencing incombination with genomic sequence-specific amplification technologies,such as the polymerase chain reaction may be utilized to facilitate therecovery of the desired genes (Mullis et al., 1986; European PatentApplication 50,424; European Patent Application. 84,796, European PatentApplication 258,017, European Patent Application. 237,362; EuropeanPatent Application. 201,184; U.S. Pat. Nos. 4,683,202; 4,582,788; and4,683,194), all of the above incorporated herein by reference.

ii. Exonuclease Resistance

Other methods that can be employed to determine the identity of anucleotide present at a polymorphic site utilize a specializedexonuclease-resistant nucleotide derivative (U.S. Pat. No. 4,656,127). Aprimer complementary to an allelic sequence immediately 3′- to thepolymorphic site is hybridized to the DNA under investigation. If thepolymorphic site on the DNA contains a nucleotide that is complementaryto the particular exonucleotide-resistant nucleotide derivative present,then that derivative will be incorporated by a polymerase onto the endof the hybridized primer. Such incorporation makes the primer resistantto exonuclease cleavage and thereby permits its detection. As theidentity of the exonucleotide-resistant derivative is known one candetermine the specific nucleotide present in the polymorphic site of theDNA.

iii. Microsequencing Methods

Several other primer-guided nucleotide incorporation procedures forassaying polymorphic sites in DNA have been described (Komher et al.,1989; Sokolov, 1990; Syvanen 1990; Kuppuswamy et al., 1991; Prezant etal., 1992; Ugozzoll et al., 1992; Nyren et al., 1993). These methodsrely on the incorporation of labeled deoxynucleotides to discriminatebetween bases at a polymorphic site. As the signal is proportional tothe number of deoxynucleotides incorporated, polymorphisms that occur inruns of the same nucleotide result in a signal that is proportional tothe length of the run (Syvanen et al., 1990).

iv. Extension in Solution

French Patent 2,650,840 and PCT Application WO91/02087 discuss asolution-based method for determining the identity of the nucleotide ofa polymorphic site. According to these methods, a primer complementaryto allelic sequences immediately 3′- to a polymorphic site is used. Theidentity of the nucleotide of that site is determined using labeleddideoxynucleotide derivatives which are incorporated at the end of theprimer if complementary to the nucleotide of the polymorphic site.

v. Genetic Bit Analysis or Solid-Phase Extension

PCT Application WO92/15712 describes a method that uses mixtures oflabeled terminators and a primer that is complementary to the sequence3′ to a polymorphic site. The labeled terminator that is incorporated iscomplementary to the nucleotide present in the polymorphic site of thetarget molecule being evaluated and is thus identified. Here the primeror the target molecule is immobilized to a solid phase.

vi. Oligonucleotide Ligation Assay (OLA)

This is another solid phase method that uses different methodology(Landegren et al., 1988). Two oligonucleotides, capable of hybridizingto abutting sequences of a single strand of a target DNA are used. Oneof these oligonucleotides is biotinylated while the other is detectablylabeled. If the precise complementary sequence is found in a targetmolecule, the oligonucleotides will hybridize such that their terminiabut, and create a ligation substrate. Ligation permits the recovery ofthe labeled oligonucleotide by using avidin. Other nucleic aciddetection assays, based on this method, combined with PCR have also beendescribed (Nickerson et al., 1990). Here, PCR is used to achieve theexponential amplification of target DNA, which is then detected usingthe OLA.

vii. Ligase/Polymerase-Mediated Genetic Bit Analysis

U.S. Pat. No. 5,952,174 describes a method that also involves twoprimers capable of hybridizing to abutting sequences of a targetmolecule. The hybridized product is formed on a solid support to whichthe target is immobilized. Here the hybridization occurs such that theprimers are separated from one another by a space of a singlenucleotide. Incubating this hybridized product in the presence of apolymerase, a ligase, and a nucleoside triphosphate mixture containingat least one deoxynucleoside triphosphate allows the ligation of anypair of abutting hybridized oligonucleotides. Addition of a ligaseresults in two events required to generate a signal, extension andligation. This provides a higher specificity and lower “noise” thanmethods using either extension or ligation alone and unlike thepolymerase-based assays, this method enhances the specificity of thepolymerase step by combining it with a second hybridization and aligation step for a signal to be attached to the solid phase.

viii. Invasive Cleavage Reactions

Invasive cleavage reactions can be used to evaluate cellular DNA for aparticular polymorphism. A technology called INVADER® employs suchreactions (e.g., de Arruda et al., 2002; Stevens et al., 2003, which areincorporated by reference). Generally, there are three nucleic acidmolecules: 1) an oligonucleotide upstream of the target site (“upstreamoligo”), 2) a probe oligonucleotide covering the target site (“probe”),and 3) a single-stranded DNA with the target site (“target”). Theupstream oligo and probe do not overlap but they contain contiguoussequences. The probe contains a donor fluorophore, such as fluoroscein,and an acceptor dye, such as Dabcyl. The nucleotide at the 3′ terminalend of the upstream oligo overlaps (“invades”) the first base pair of aprobe-target duplex. Then the probe is cleaved by a structure-specific5′ nuclease causing separation of the fluorophore/quencher pair, whichincreases the amount of fluorescence that can be detected. See Lu et al.(2004). In some cases, the assay is conducted on a solid-surface or inan array format.

III. PREDICTING AND DIAGNOSING MULTIPLE SCLEROSIS

A. Multiple Sclerosis

Multiple Sclerosis (MS) is one of the most common diseases of thecentral nervous system (brain and spinal cord). It is an inflammatorycondition associated with demyelination, or loss of the myelin sheath.Myelin, a fatty material that insulates nerves, acts as insulator inallowing nerves to transmit impulses from one point to another. In MS,the loss of myelin is accompanied by a disruption in the ability of thenerves to conduct electrical impulses to and from the brain and thisproduces the various symptoms of MS, such as impairments in vision,muscle coordination, strength, sensation, speech and swallowing, bladdercontrol, sexuality and cognitive function. The plaques or lesions wheremyelin is lost appear as hardened, scar-like areas. These scars appearat different times and in different areas of the brain and spinal cord,hence the term “multiple” sclerosis, literally meaning many scars.

Currently, there is no single laboratory test, symptom, or physicalfinding that provides a conclusive diagnosis of MS. To complicatematters, symptoms of MS can easily be confused with a wide variety ofother diseases such as acute disseminated encephalomyelitis, Lymedisease, HIV-associated myelopathy, HTLV-1-associated myelopathy,neurosyphilis, progressive multifocal leukoencephalopathy, systemiclupus erythematosus, polyarteritis nodosa, Sjögren's syndrome, Behçet'sdisease, sarcoidosis, paraneoplastic syndromes, subacute combineddegeneration of cord, subacute myelo-optic neuropathy,adrenomyeloneuropathy, spinocerebellar syndromes, hereditary spasticparaparesis/primary lateral sclerosis, strokes, tumors, arteriovenousmalformations, arachnoid cysts, Arnold-Chiari malformations, andcervical spondylosis. Consequently, the diagnosis of MS must be made bya process that demonstrates findings that are consistent with MS, andalso rules out other causes.

Generally, diagnosis of MS relies on two criteria. First, there musthave been two attacks at least one month apart. An attack, also known asan exacerbation, flare, or relapse, is a sudden appearance of orworsening of an MS symptom or symptoms which lasts at least 24 hours.Second, there must be more than one area of damage to central nervoussystem myelin sheath. Damage to sheath must have occurred at more thanone point in time and not have been caused by any other disease that cancause demyelination or similar neurologic symptoms. MRI (magneticresonance imaging) currently is the preferred method of imaging thebrain to detect the presence of plaques or scarring caused by MS.

The diagnosis of MS cannot be made, however, solely on the basis of MRI.Other diseases can cause comparable lesions in the brain that resemblethose caused by MS. Furthermore, the appearance of brain lesions by MRIcan be quite heterogeneous in different patients, even resembling brainor spinal cord tumors in some. In addition, a normal MRI scan does notrule out a diagnosis of MS, as a small number of patients with confirmedMS do not show any lesions in the brain on MRI. These individuals oftenhave spinal cord lesions or lesions which cannot be detected by MRI. Asa result, it is critical that a thorough clinical exam also include apatient history and functional testing. This should cover mental,emotional, and language functions, movement and coordination, vision,balance, and the functions of the five senses. Sex, birthplace, familyhistory, and age of the person when symptoms first began are alsoimportant considerations. Other tests, including evoked potentials(electrical diagnostic studies that may reveal delays in central nervoussystem conduction times), cerebrospinal fluid (seeking the presence ofclonally-expanded immunoglobulin genes, referred to as oligoclonalbands), and blood (to rule out other causes), may be required in certaincases.

B. Samples and Preparation

The present invention contemplates the identification of VH4 sequencesfrom B cells obtained from any sample (fluid or tissue) that wouldcontain such cells. In particular, the present invention will rely onperipheral blood as a source of B cells, given the ease of obtention andthe plentiful nature of B cells. In addition, given the CNS implicationsof MS, cerebrospinal fluid provides another potential source of B cellsfor analysis. Methods for separating and analyzing nucleic acids areprovided above.

C. Therapy and Prophylaxis

It may be that, on the basis of the diagnosis or prediction provided bythe methods described herein, one will wish to begin, end or modify atherapeutic regimen. In particular, subjects diagnosed as having or atrisk of developing MS may be started on a therapeutic regimen. Theprimary aims of therapy are returning function after an attack,preventing new attacks, and preventing disability. As with any medicaltreatment, medications used in the management of MS have several adverseeffects, and many possible therapies are still under investigation.

During symptomatic attacks, administration of high doses of intravenouscorticosteroids, such as methylprednisolone, is the routine therapy foracute relapses. The aim of this kind of treatment is to end the attacksooner and leave fewer lasting deficits in the patient. Althoughgenerally effective in the short term for relieving symptoms,corticosteroid treatments do not appear to have a significant impact onlong-term recovery. Potential side effects include osteoporosis andimpaired memory, the latter being reversible.

The earliest clinical presentation of relapsing-remitting MS (RRMS) isthe clinically isolated syndrome (CIS). Several studies have shown thattreatment with interferons during an initial attack can decrease thechance that a patient will develop MS. As of 2007, six disease-modifyingtreatments have been approved by regulatory agencies of differentcountries for relapsing-remitting MS. Three are interferons: twoformulations of interferon beta-1a (trade names Avonex and Rebif) andone of interferon beta-1b (U.S. trade name Betaseron™, in Europe andJapan Betaferon). A fourth medication is glatiramer acetate (Copaxone™).The fifth medication, mitoxantrone, is an immunosuppressant also used incancer chemotherapy, is approved only in the USA and largely for SPMS.Finally, the sixth is natalizumab (marketed as Tysabri™). All sixmedications are modestly effective at decreasing the number of attacksand slowing progression to disability, although they differ in theirefficacy rate and studies of their long-term effects are still lacking.Comparisons between immunomodulators (all but mitoxantrone) show thatthe most effective is natalizumab, both in terms of relapse ratereduction and halting disability progression; it has also been shown toreduce the severity of MS. Mitoxantrone may be the most effective ofthem all; however, it is generally considered not as a long-term therapyas its use is limited by severe cardiotoxicity.

The interferons and glatiramer acetate are delivered by frequentinjections, varying from once-per-day for glatiramer acetate toonce-per-week (but intra-muscular) for Avonex. Natalizumab andmitoxantrone are given by IV infusion at monthly intervals. Treatment ofprogressive MS is more difficult than relapsing-remitting MS.Mitoxantrone has shown positive effects in patients with a secondaryprogressive and progressive relapsing courses. It is moderatelyeffective in reducing the progression of the disease and the frequencyof relapses in patients in short-term follow-up. On the other hand notreatment has been proven to modify the course of primary progressiveMS.

Disease-modifying treatments only reduce the progression rate of thedisease but do not stop it. As multiple sclerosis progresses, thesymptomatology tends to increase. The disease is associated with avariety of symptoms and functional deficits that result in a range ofprogressive impairments and handicap. Management of these deficits istherefore very important. Both drug therapy and neurorehabilitation haveshown to ease the burden of some symptoms, even though neither influencedisease progression. As for any patient with neurologic deficits, amultidisciplinary approach is key to limiting and overcoming disability;however there are particular difficulties in specifying a ‘core team’because people with MS may need help from almost any health professionor service at some point. Similarly for each symptom there are differenttreatment options. Treatments should therefore be individualizeddepending both on the patient and the physician.

The present invention also contemplates the use of novel therapeuticagents—antibodies or peptides/peptoids that bind to the altered VH4genes described herein—to treat SLE. VH4-antibody therapeutics can beprepared and screened for reactivity using well known techniques.Peptides and peptiods that act as “mimotopes,” or epitope-mimickingstructures can be administered and used to sequester the VH4 productsaway from pathologic interactions. See Reimer & Jensen-Jarolim (2007).

IV. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Materials and Methods

Patient Description and Database Generation.

Cerebrospinal fluid and peripheral blood were collected from elevenrelapsing remitting MS patients at UT Southwestern Medical Center, inaccordance with the UT Southwestern Institutional Review Board. Briefpatient descriptions are summarized in Table 7. This database includespatients whose sequences have been analyzed elsewhere (Monson et al.,2005; Harp et al., 2007). Sequences obtained from CSF-derived B cellrepertoires from these patients were analyzed using Sequencher 4.5 (GeneCodes Corporation, Ann Arbor, Mich.). Differences from the IgBlast(NCBI; world wide web at ncbi.nlm.nih.gov/igblast) were evaluated, butno changes in gene usage were noted. Sequences with more than 4mutations (less than 98% germline) were designated as the MS memory(mMSCSF) subdatabase (Brezinschek et al., 1998; Damle et al., 1999;Hamblin et al., 1999). Two RRMS patients were collected under theUniversity of Colorado School of Medicine Institutional Review Board(Ritchie et al., 2004). Brief patient descriptions are included in Table7. B cell repertoire summaries from these patients was published inRitchie et al. (2004), and analyzed using DNASIS Max software located atthe V Base Sequence directory (world wide web at mrc-cpe.cam.ac.uk). TheHCPB antibody database has been used in multiple studies (Brezinschek etal., 1997; Brezinschek et al., 1998; Dorner et al., 1997; Dorner et al.,1998a; Dorner et al., 1998b; Dorner et al., 1998c; Farner et al., 1999;Hansen et al., 2000; Monson et al., 2000). The MSPB database wascollected from the peripheral blood of three relapsing remitting MSpatients (see Table 7 for brief patient summaries) at the time of CSFsampling. Peripheral blood lymphocytes were isolated by centrifugationin the presence of a Ficoll gradient. Similarly to HCPB, the inventorseparated those sequences that contained more than 4 mutations (lessthan 98% germline sequence), which would represent peripheral memory Bcells (Brezinschek et al., 1998; Damle et al., 1999; Hamblin et al.,1999) (memory MSPB or mMSPB).

The patients used to generate the class-switched IgG₊CD27₊ database aredescribed in Tian et al. (2007) and were collected under the VanderbiltUniversity Medical Center Institutional Review Board. The sequences werere-confirmed by the inventor's laboratory using IgBlast for gene usage,mutational number, and mutational codon location and type (replacementor silent). These sequences were combined with sequences from the HCPBdatabase described above that contained more than 4 mutations (less than98% germline sequence), which would represent peripheral memory B cells.The HCPB memory (mHCPB) sub-database and the class-switched IgG B celldatabase (Tian et al., 2007) did not differ in gene family usage, butdid differ in MF.

-   -   One patient with Sjögren's syndrome was used; the data from this        patient was published in (Hansen et al., 2003), and are listed        in Table 6.    -   One patient with systemic lupus erythematosus (SLE) was used;        the data from this patient was published in (Domer et al.,        1999), and were analyzed using GeneWorks (IntelliGenetics,        Mountain View, Calif.) and Sequencher (Gene Codes, Ann Arbor,        Mich.). A patient summary can be found in Table 8.    -   Two patients with other neurological diseases (OND) were        collected from UT Southwestern Medical Center, in accordance        with UT Southwestern Institutional Review Board. The obtained        sequences were analyzed using Sequencher 4.5 (Gene Codes        Corporation, Ann Arbor, Mich.). The patients are listed in Table        8.    -   Two patients were obtained that had experienced a clinically        isolated event;

the diagnostic lumbar puncture was used as the CSF B cell sort and werecollected from UT Southwestern Medical Center, in accordance with UTSouthwestern Institutional Review Board.

The obtained sequences were analyzed using Sequencher 4.5 (Gene Codes,Ann Arbor, Mich.) for CIS 132, and the sequences from CIS429 wereanalyzed using IgBlast. An additional CIS patient was collected underthe University of Colorado School of Medicine Institutional ReviewBoard; the data from this patient was published in Ritchie et al.(2004), and analyzed using DNASIS Max software using V Base Sequencedirectory (world wide web at mrccpe.cam.ac.uk). The patients included inthese analyses are listed in Table 8.

B Cell Sorting, Primer Extension Preamplification, and Heavy ChainRearrangement Amplification.

These methods were carried out as previously described (Farner et al.,1999; Brezinschek et al., 1995; Foster et al., 1999). Clonally expandedB cells were defined as those VH rearrangements being represented two ormore times in the repertoire. Clones were determined by similar VH andJH usage, followed by CDR3 length and composition; mutations were thencompared to ensure clonality. In normalized analyses, the clones wereonly counted a single time, regardless of the number of rearrangementsin the repertoire. Two additional MS CD19₊ rearrangement sequences usedin VH family, VH4 gene, JH usage, overall MF, and CDR3 length numberswere obtained from (Ritchie et al., 2004).

Mutational Analyses.

VH read length was defined as codons 31 through 95 (CDR1 and 2, FR 2 and3). The 3′ end of the VH gene was defined as codon 95, or as long as thegermline variable sequence was present. Mutational frequency wasdetermined as the number of mutations as related by germline VHdatabases (Sequencher 4.5 and IgBlast) and divided by the total readlength, not including FR1 in either of these figures. Mutationalfrequency in CDR or FR was done in the same manner, with the number ofmutations in the regions (CDR1 and CDR2; FR2 and FR3) divided by thetotal number of nucleotides in that region.

Targeting to DGYW/WRCH motifs was evaluated by mutational frequency in amotif; this number was generated using the number of mutations in amotif divided by the total number of nucleotides in DGYW/WRCH motifs(done by the number of nucleotides in a motif of each gene multiplied bythe number of times this gene was used in the repertoire). Only CDR1,FR2, CDR2, and FR3 were included in this analysis.

Mutational position frequency was calculated as the number ofreplacement mutations between codons 24 to 95 at each codon divided bythe total number of replacement mutations. Codons 24 to 31 of FR1 wereincluded in this analysis because most of the sequences did includethese locations, and thus might be biased, but conservatively. Codondomains were defined by Kabat (1987), and codon numbers were defined byTomlinson in V-base (vbase.mrc-cpe.cam.ac.uk).

Each mutation in CDR1, FR2, CDR2, and FR3 was counted once in mutationalfrequency and CDR targeting analyses, and each codon only counted oncefor replacement: silent ratios.

CDR3 Length.

CDR3 length was considered from the end of VH, including the D segmentto the beginning of the JH segment, ending at codon 102, as defined byKabat (1987).

VH4 Structure.

A human VH4-30.4 antibody structure was obtained from the Protein DataBank, Chemistry Department, Brookhaven National Laboratory, Upton, N.Y.11973 (world wide web at rcsb.org) under the identification moniker1MCO, and adapted using the RasMol program (RasMac v2.6 available atmc2.cchem.berkeley.edu/Rasmol/Sayle and Milner-White, 1995)). Thestructure was described in (Guddat et al., 1993), and deposited in thedata bank by the authors. The adaptations made were to show only thevariable regions (including VDJ), and to highlight the codons includedin the signature as described in the figure legends.

Statistical Analysis.

Family and VH4 gene usages were compared using chi square analysis. CDR3lengths were analyzed by ANOVA analysis, using the Kruskal-Wallis test.Mutational position was compared between MS and HCPB using the Goodnessof Fit test.

Example 2 Results

MS Patient CSF B Cells have an Unusually High Frequency of VH4 FamilyUsage.

In order to address our hypothesis that the VH4 family used in the MSCSFwill be dysregulated, the inventor first analyzed whether the VH4 familywas overrepresented in our CSF-derived B cell antibody databaseconsisting of 405 sequences from 13 MS patients (patient descriptions inTable 7) and compared it to healthy control B cell repertoires (patientdescriptions in Table 8) (Brezinschek et al., Brezinschek et al., 1998;Dorner et al., 1997; Dorner et al., 1998a; Dorner et al., 1998b; Dorneret al., 1998c; Farner et al., 1999; Hansen et al., 2000; Monson et al.,2000). As previously established, the majority of B cells from healthycontrol peripheral blood (HCPB) most often utilize VH3 family genes(61.1%) to generate their antibody repertoire, followed by VH4 familygenes (18.5%) and VH1 family genes (13.7%) (FIG. 1A) (Brezinschek etal., 1995). VH2 and VH5 family genes are rarely rearranged in B cellsfrom HCPB (2.2% and 4% respectively) (Brezinschek et al., 1995).However, HCPB is composed mostly of naïve B cells, which likely have adifferent family usage signature in comparison to CSF B cells, which arecomposed mostly of memory B cells (Harp et al., 2007; Cepok et al.,2005). Thus, we generated a sub-database of only those sequences fromthe HCPB database that would be categorized as memory B cells based onhomology (<98%) as previously defined by us and others (Brezinschek etal., 1998; Damle et al., 1999; Hamblin et al., 1999). The inventor alsocombined our HCPB memory B cell sub-database with a B cell databasegenerated from post-switch memory B cells (CD27₊IgG₊) (Tian et al.,2007). These 2 databases did not differ statistically from each other inVH1, VH2, VH3 or VH4 family usage. Comparison of this memorysub-database (mHCPB) to the full database (HCPB) revealed no significantchanges in VH family usage, such that VH3 family genes constituted themajority of VH family usage in the repertoire (compare 61.1% in HCPB to61.5% in mHCPB, p>0.14). VH1 and VH4 family usage was also comparablebetween the two databases. The frequency of VH4 family usage is alsostatistically similar to peripheral memory B cells from MS patients(mMSPB), even though it appears different (FIG. 1A) (compare 16.4% mHCPBto 22.3% mMSPB, p>0.26).

In contrast to the mHCPB database, the CSF B cell database from MSpatients (MSCSF) had a significant decrease in VH3 family usage (compare61.5% in mHCPB to 31.4% in MSCSF, p<0.001), and an increased usage ofthe VH4 family (compare 16.6% in mHCPB to 35.8% in MSCSF, p<0.001) andthe VH1 family (compare 11.1% in mHCPB to 25.2% in MSCSF, p<0.001)(FIGS. 1A and 1B). Overrepresentation of VH4 usage was maintained evenwhen each clone was counted once in the repertoire database (normalized;FIG. 1B). Plasma B cell repertoires (identified by CD138 expression)from MS patient CSF reported by others (Ritchie et al., 2004; Owens etal., 2007) also demonstrated a pronounced skewing to VH4 family usage(66.7%), but did not show the increased VH1 family usage seen in theCD19+ B cell population (compare 2.3% in CD138₊ to 25.2% in CD19₊,p<0.001) (FIG. 1B).

Patients with Clinically Isolated Syndrome (CIS) are considered “atrisk” to develop MS, but did not demonstrate VH4 prevalence observed inthe MSCSF database since VH4 frequency in the CISCSF was statisticallyless than the MSCSF (compare 22.0% in CISCSF to 35.8% in MSCSF, p<0.01)(FIG. 1C), but similar to HCPB (compare 22.0% in CISCSF to 21.8% inHCPB, p=0.96) even when only the patients that progressed to CDMS wereconsidered. The CISCSF_(VH4) frequency is similar to that of the HCPB,mHCPB, MSPB, mMSPB, and ONDCSF. Interestingly, a previously reported CISpatient did have this increased frequency of VH4 family usage in theCSF-derived plasma cell repertoire (Ritchie et al., 2004), even thoughit is has yet to be demonstrated in the same patient's CSF-derived CD19₊population (compare 25.0% in CISCSF or mCISCSF to 42.1% in CIS CD138,p<0.005) (FIG. 1C).

To determine if overrepresentation of VH4 family usage was unique to theMS patients, or could be observed in other patients with autoimmunediseases mediated by humoral immunity, the inventor compared thesignature of VH family usage from the peripheral blood of two other Bcell autoimmune diseases, SLE (Hansen et al., 2000; Dorner et al., 1999)and Sjögren's Syndrome (Hansen et al., 2003), as well as two CSF-derivedB cell repertoires from patients with other neurological diseases (OND)(Monson et al., 2005; Harp et al., 2007) to the MSCSF database. Asindicated in FIG. 1D, B cell repertoires from Sjögren's or SLE patientsdid not utilize the VH4 family as extensively as CSF-derived B cellsfrom MS patients (FIG. 1B). In addition, neither OND patient utilizedthe VH4 family more extensively than expected, regardless of theinflammatory nature of the patient's disease (FIG. 1D).

MS Patient CSF B Cell Database Reveals No Restriction in Individual VH4Gene Usage.

The increase of VH4 family usage in the MSCSF database in comparison tomHCPB and patients with other B cell mediated autoimmune diseases couldbe attributed to an increased usage frequency of all nine individualheavy chain genes that comprise the VH4 family, or preferential use ofone or more of the VH4 family genes. To differentiate between these twopossibilities, we compared the frequency usage of the 9 individual genesthat comprise the VH4 family in the MSCSF database to the mHCPB database(Table 1) and repertoires from patients with other B cell mediatedautoimmune diseases (Table 2). All of the VH4 genes were used similarlyin HCPB and mHCPB, with the exception of 4-04, which is observed moreoften in mHCPB than in the inclusive repertoire (compare 9% in the HCPBto 29% in mHCPB, p<0.03) (Table 1). MSPB and HCPB also had similar VH4gene frequencies, as did mMSPB and mHCPB. MSCSF usage of the VH4 geneswas also the same as in mMSPB, but resembled HCPB instead of mHCPB inVH4-04 usage (6% in MSCSF compared to 9% in HCPB, p=0.41, or 29% inmHCPB, p<0.001) (Table 1). These observations were maintained even whenthe repertoires were normalized so that each clone was represented oncein the MSCSF database. Interestingly, VH4 individual gene usagefrequency was similar in MSCSF, SLE and Sjögren's, with the exception ofVH4-34, which was used more extensively in both SLE and Sjögren'scompared to MSCSF (Table 2).

J Segment Usage not Biased in MSCSF_(VH4) Populations, but is inMSPB_(VH4).

The extensive VH4 family usage by the MSCSF database prompted analysisof the J segment usage within the VH4-expressing B cell databases.Autoreactive B cells in peripheral germinal centers are known to utilizeJH6 segments more frequently (Zheng et al., 2004), so the inventorreasoned that VH4-expressing B cells from the CSF of MS patients mayalso be enriched for JH6 usage. JH4 is the most common J segment used inthe HCPB B cell repertoire database as described by the inventor (FIG. 6and (Monson et al., 2005; Harp et al., 2007) and others (Brezinschek etal., 1995), even when only those B cells expressing VH4 family genes areconsidered (FIG. 2, HCPB_(VH4)=57.7%). The subdatabase consisting ofonly those memory B cells expressing VH4 family genes also utilized theJH4 segment most frequently (mHCPB_(VH4)=57.1%). In contrast, themMSPB_(VH4) database utilized JH6 segments more frequently than mHCPB(compare 58.8% mMSPB_(VH4) to 11.4% mHCPB_(VH4), p<0.001). Curiously,this enrichment of JH6 utilization by VH4 expressing B cells observed inMSPB was not observed in MSCSF_(VH4) (compare 40.0% MSPB_(VH4) to 14.6%MSCSF_(VH4), p<0.001).

MSCSF_(VH4) Cells have Normal CDR3 Length, but MSPB_(VH4) do not.

Longer CDR3 lengths have been associated with dysregulation andautoimmunity (Wardemann et al., 2003), so the inventor reasoned thatVH4-expressing B cells from MSCSF may have longer CDR3 lengths than theVH4-expressing B cells from HCPB. CDR3 lengths are typically comparedamong groups by calculating the average length (FIG. 3A); however,different distributions can result in a similar average, so distributionranges are also useful for comparison (FIG. 3B). HCPB as a whole has aCDR3 length average of 13.1 amino acids, and the VH4 subset has anaverage of 13.4 amino acids (p>0.05). The mHCPB_(VH4) has a mean of 12.7and the MSCSF_(VH4), though longer (14.1 amino acids), did not differstatistically from either of these (p>0.05).

In contrast, the CDR3 distribution ranges did differ (FIG. 3B),especially when MSPB or mMSPB were compared to HCPB. For example,MSPB_(VH4) had 22.5% of its sequences in the longest range of >20 aminoacids, and HCPB_(VH4) had 4.0% of its sequences in the longest rangeof >20 amino acids (p<0.003). However, CDR3 lengths>20 amino acids wereat a similar frequency in MSCSF and mMSCSF compared to HCPB (9.0% and10.3% compared to 4.0%, p=0.175 and p=0.111 respectively). This dataindicated that MSPB and mMSPB tended towards longer CDR3 lengths morereadily than the other groups (including MSCSF), although all groupstypically had CDR3 lengths in the range of 10 to 14 amino acids.

MSCSF_(VH4) have an Increased Mutational Frequency in Comparison toHCPB_(VH4).

Previously, the inventor established that the MSCSF B cell database hadan enhanced mutational frequency (MF) in comparison to HCPB (Monson etal., 2005. The inventor hypothesized that VH4-expressing CSF-derived Bcells would also have a higher mutation frequency than what is observedin the population as a whole, especially considering the increasedfrequency of VH4-expressing B cells, suggesting local expansion of thispopulation. In order to test this hypothesis, the inventor compared themutational frequencies of the B cell databases as a whole to themutational frequencies of the sub-database of only those B cellsexpressing VH4 genes (Table 3 and FIG. 4). As expected, the inclusiveHCPB repertoire has a mutational frequency of 2.3% because the majorityof the B cells in this compartment are naïve and, as expected, thememory B cell subpopulation of this database (mHCPB) had a much higherMF (compare 2.3% in HCPB to 5.8% in mHCPB, p<0.001). Interestingly, theVH4-expressing B cell subpopulation of this database (HCPB_(VH4)) had aMF of 2.0%, which was significantly less than the MF of the overallrepertoire without VH4 (compare 2.0% in HCPB_(VH4) to 2.3% inHCPB_(All-VH4), p<0.02). In contrast, the MSPB_(VH4) and the MSCSF_(VH4)subdatabases had MFs that were statistically greater than the MF of theoverall MSPB and MSCSF databases (compare 2.9% in MSPB_(VH4) to 1.7% inMSPB (p<0.001) and 6.0% in MSCSF_(VH4) to 5.0% in MSCSF (p<0.001)). Thissame pattern was observed when only memory B cells were considered, andis due to the enrichment of CSF B cells with more than 5 mutations perrearrangement (FIG. 4). B cell repertoires from the CIS, Sjögren's, orSLE patient populations were not analyzed in this manner because of thelow frequency of VH4 expressing B cells in those repertoires.

MSCSF_(VH4) Mutational Characteristics Retain Targeting to CDR andDGYW/WRCH Motifs.

Mutational characteristics of the antibody variable region can confirmwhether appropriate targeting of mutations that are associated withantigenic selection occur within the context of a classic germinalcenter (Harp et al., 2007). The MSCSF database maintains typicalgerminal center features including targeting to CDRs and particularmotifs within the CDRs (Harp et al., 2007). CSF derived B cell clonesfrom MS patients have more atypical features (Monson et al., 2005),suggesting that some clonally expanded B cells in the CSF are notselected in the context of a classical germinal center. If the B cellsexpressing VH4 genes are enriched for self-reactive B cells, and thesecells are being driven by antigen in the CNS, the VH4 cells may havediminished mutational targeting characteristics rather than thepunctuated targeting of mutations associated with classically selectedgerminal center B cells. To evaluate this, the inventor categorized4,182 mutations in the MSCSF B cell database and 1,815 mutations in theMSCSF_(VH4) sub-database according to their regional location, aminoacid position, whether the mutation resulted in an amino acid change(replacement) or not (silent), and whether the mutation occurred withina motif known to be targeted by the mutational machinery (Rogozin andDiaz, 2004). The combination of these traits indicate whether or not a Bcell or population of B cells have been selected in the context of aclassical germinal center.

Mutational Targeting Measured by MF in MSCSF_(VH4) CDR is Preserved.

Since the CDR is comprised of fewer nucleotides than the FR, mutationalfrequencies in these regions are a more objective method of evaluatingtargeting than the percentage of mutations in these regions. As onewould expect in a typical germinal center reaction, the MSCSF CDRs havea much higher MF than FRs when the repertoire is considered as a whole(8.0 to 3.1, p<0.001), or when only VH4 expressing CSF-derived B cellsare considered (8.8 to 3.9, p<0.001) (Table 3). When only memory MSCSF Bcells are considered, the CDR MF is still much higher than the FR MFwhen the repertoire is considered as a whole (11.8 to 4.6, p<0.001), orwhen only the mMSCSF_(VH4) sub-database is considered (11.7 to 5.7,p<0.004). This implies that targeting mutations to CDR is preserved inthe VH4 expressing B cells from MSCSF.

Replacement:Silent Ratios are Normal in MSCSF_(VH4).

It is well established that replacement mutations within CDRs arefavorable as they influence antigen affinity, whilst replacementmutations within FRs are unfavorable as they can affect antibodystructure (Kirkham and Schroeder, 1994; Vargas-Madrazo et al., 1994;Both et al., 1990; Tanaka and Nei, 1989). An R:S ratio of 2.9 isconsidered random (Shlomchik et al., 1987), less than this number isconservation of sequence, and a ratio greater than 2.9 indicatesdiversification (Shlomchik et al., 1987). As in the HCPB_(VH4), theMSCSF_(VH4) B cells had significant sequence variation in the CDR, butpreservation of sequence in the FR(HCPB_(VH4) CDR 6.6, FR 1.3;MSCSF_(VH4) CDR 4.4, FR 1.2) (Table 4).

Targeting to DGYW/WRCH Motifs is Preserved in MSCSF_(VH4).

Somatic hypermutation occurring in the context of a classical germinalcenter is predominantly targeted to DGYW/WRCH motifs within variableimmunoglobulin genes (Rogozin and Diaz, 2004). If VH4 expressing B cellsundergo antigen driven selection in the context of a classical germinalcenter, then targeting to these motifs should be preserved. In order todetermine whether appropriate targeting to DGYW/WRCH (abbreviated “DW”)motifs occurred in the MSCSF VH4 sub-database in comparison to the VH4sub-database of the control groups, MFs in the motifs were determined.HCPB_(VH4) B cells had a MF within DW motifs of 3.1%, while themHCPB_(VH4) B cells had a MF of 9.4% (p<0.001)(Table 4). MSCSF_(VH4) Bcells had a MF of 9.0% within DW motifs, which was statistically greaterthan what was observed in HCPB_(VH4) B cells (p<0.001), equivalent towhat was observed in mHCPB (p>0.05).

Mutation Position Analysis Reveals an MS-Specific Signature in VH4Expressing CSF-Derived B Cells.

Analysis of individual codons' mutation frequencies may possibly reveala pattern of replacement mutations in the VH4 genes that is unique tothe MSCSF database. In order to test this, the mutational frequency ateach codon within the MSCSF_(VH4) B cell database was determined andcompared to the frequency of random mutation (1.5%) such that any codonwith a MF statistically greater than 1.5% was identified as a “hot” spot(Dorner et al., 1997) (Table 5). Previous analysis had identified codonpositions 30, 31, 50, 55, 56, 78, 89 and 94 as “hot spots” forreplacement mutations in the overall HCPB heavy chain repertoire (Dorneret al., 1997). The VH4 subdatabase of the HCPB database (HCPB_(VH4))includes two of these original hotspots (codons 30 and 56), and 3additional hotspots for replacement mutations (codons 52, 68, and 81),which were marked as VH4 family biased mutational hot spots (Table 5).Interestingly, one of the three VH4 family biased mutational hotspots isnot included within a DGYW/WRCH motif (codon 52), but it has amutational frequency significantly greater than the random frequency(compare 4.9% to the random frequency of 1.5%, p<0.001).

Five additional positions (codons 31B, 40, 57, 60, and 69) wereidentified as replacement hot spots in the MSCSF_(VH4) database thatwere not replacement hot spots in either the HCPB_(VH4) database oroverall HCPB databases (Table 18), and are thus specific forMSCSF-derived B cells expressing VH4 antibody genes. Of these MSspecific hotspots, 31B is most impressive, demonstrating a 7-foldincrease in mutation accumulation in comparison to HCPB. This codon ispresent in only four of the nine VH4 genes (4-30, 4-31, 4-39, 4-61), andis mutated in 69% of B cells utilizing VH4-61, 46% of B cells utilizingVH4-39, 35% of B cells utilizing VH4-30, and 14% of B cells utilizingVH4-31 (data not shown). Interestingly, VH4-34, the VH4 variable geneassociated with autoreactivity in the periphery of healthy controls andSLE patients (Pugh-Bernard et al., 2001; Zheng et al., 2004; Mockridgeet al., 2004; Voswinkel et al., 1997), does not contain this codon.Codons 30 and 68 are “cold” spots, in that the MSCSF_(VH4) B cells hadreplacement mutations at these positions significantly less frequentlythan in HCPB_(VH4) B cells (Table 5). Codon 52 in the MSCSF_(VH4)database also had an MF that was less than the HCPB_(VH4) database, butstill significantly greater than the random frequency. These “hot” and“cold” spots together include 11 codons, and represent 27.8% of themutations in the MSCSF_(VH4) database (Table 6). These significantchanges in codon mutation frequency in the MSCSF_(VH4) databaseconstitute a footprint of mutations that is unique to MS.

TABLE 1 VH4 individual gene usage in peripheral blood HCPB MSPB MSCSFGenes HCPB Memory MSPB Memory MSCSF Memory 4-04 7 (9%)^(4,5) 9 (26%) 4(10%) 2 (14%)⁷ 9 (6%)⁵ 3 (3%)^(5,6) 4-28 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0(0%) 0 (0%) 4-30¹ 5 (7%)⁷ 2 (6%) 5 (13%) 2 (14%) 26 (18%) 23 (21%)⁵ 4-314 (5%) 0 (0%) 3 (8%) 1 (7%) 7 (5%) 6 (6%) 4-34 13 (17%) 3 (9%) 4 (10%) 1(7%) 8 (6%) 5 (5%) 4-39 16 (21%) 6 (17%) 9 (23%) 1 (7%) 35 (24%) 23(22%) 4-59 24 (32%) 11 (31%) 11 (28%) 5 (36%) 41 (28%) 29 (27%) 4-61 3(4%) 1 (3%) 2 (5%) 1 (7%) 13 (9%) 12 (11%) 4-B 4 (5%) 3 (9%) 2 (5%) 1(7%) 6 (4%) 3 (3%) VH4 N² 76 35 40 14 145 104 Total N³ 349 205 226 62405 273 ¹Includes all sub-genes (4-30.1, 4-30.2, and 4-30.4) ²Number ofproductive VH4 sequences analyzed in each group ³Number of productive VHsequences overall ⁴Absolute number (% of total VH4 sequences) ⁵Differentfrom HCPB memory ⁶Different from MSPB memory ⁷Different from MSCSFmemory

TABLE 2 VH4 individual gene usage in Sjögren's and SLE B cell autoimmunediseases Compared to MSCSF Genes Sjögren's SLE Sjögren's SLE 4-04  3(14%)⁴ 0 (0%) = = 4-28  0 (0%) 0 (0%) NA⁵ NA⁵ 4-30¹  2 (10%) 1 (33%) = =4-31  0 (0%) 0 (0%) = = 4-34  5 (24%) 2 (67%)

p < 0.004 p < 0.002 4-39  0 (0%) 0 (0%)

= p < 0.02  4-59  8 (38%) 0 (0%) = = 4-61  3 (14%) 0 (0%) = = 4-b  0(0%) 0 (0%) = = VH4 N² 21 3 Total N³ 107 41 ¹Includes all sub-genes(4-30.1, 4-30.2, and 4-30.4) ²Number of productive VH4 sequencesanalyzed in each group ³Number of productive VH sequences overall⁴Absolute number (% of total VH4 sequences) ⁵None of the groups had anyVH4-28 genes NA = not applicable

TABLE 3 Mutational Frequency in B cell Repertoires Total MF All CDR vsFR VH4 CDR vs FR All vs VH4 Total CDR FR p-value Total CDR FR p-valuep-value All B cells HCPB 2.3^(1,2,3) 3.5^(1,2,3) 1.7^(1,2,3) p < 0.0012.0^(1,2,3) 2.4^(1,2,3) 1.9^(1,2,3) p < 0.04 <0.02⁴ MSPB 1.7^(1,2,3)3.3^(1,2,3) 1.0^(1,2,3) p < 0.001 2.9^(1,2,3) 5.2^(1,2,3) 1.9^(1,2,3) p< 0.001 <0.001⁴ MSCSF 5.0^(1,2,3) 8.0^(1,2,3) 3.1^(1,3) p < 0.001 6.0³8.8^(2,3) 3.9^(1,3) p < 0.001 <0.001⁴ Memory B cells only mHCPB 5.8³9.4³ 4.0^(2,3) p < 0.001 6.1³ 7.7^(2,3) 5.3² p < 0.004 NS mMSPB 5.6³10.5 3.3^(1,3) p < 0.001 6.3³ 11.5¹ 3.9¹ p < 0.001 <0.02⁴ mMSCSF6.6^(1,2) 11.8^(1,2) 4.6^(1,2) p < 0.001 7.3^(1,2) 11.7¹ 5.7² p < 0.001<0.001⁴ ¹Different from HCPB memory (p ≦ 0.05) ²Different from MSPBmemory (p ≦ 0.05) ³Different from MSCSF memory (p ≦ 0.05) ⁴Comparing Allwithout VH4 to VH4 NS = not significant

TABLE 4 VH4 Mutational Characteristics HCPB mHCPB⁵ CSPB⁵ MSCSF R:S Ratio6.6³ 6.9³ 3.0^(1,2) 4.4 CDR R:S Ratio FR 1.3 1.5 1.8 1.2³ DW Motifs3.1%^(2,3,4) 9.4%¹ 15.1%^(1,2,4) 9.0%^(1,3) MF Total DW Motifs2.5%^(2,3,4) 7.7%¹ 15.7%^(1,2) 10.4%^(1,2,3) MF CDR DW Motifs3.6%^(2,3,4) 10.8%¹ 14.6%^(1,2) 7.8%^(1,2,3) MF FR ¹Different from HCPBVH4 (p ≦ 0.05) ²Different from mHCPB VH4 (p ≦ 0.05) ³Different from CSPBVH4 (p ≦ 0.05) ⁴Different from MSCSF VH4 (p ≦ 0.05) ⁵mHCPB separatedinto those defined by MF >2% (mHCPB) and those defined by IgG⁺CD27⁺expression (CSPB)

TABLE 5 VH4 R Mutational Frequencies at Codon Hot Spots DGYW MSCSF toCodon Location² Motif HCPB MSCSF HCPB 30¹ FR1 Y 3.8% 1.9%

p < 0.005 31B CDR1 Y 0.5% 3.5%

p < 0.001 40 FR2 Y 1.1% 2.6%

p < 0.001 52 CDR2 N 4.9% 2.7%

p < 0.005 56¹ CDR2 Y 2.8% 5.4%

p < 0.001 57 CDR2 Y/N³ 1.1% 2.0%

p < 0.01 60 CDR2 Y 1.1% 2.4%

p < 0.001 68 FR3 N 2.2% 1.2%

p < 0.05 69 FR3 N 1.1% 2.0%

p < 0.01 81 FR3 Y 2.7% 4.6%

p < 0.001 89¹ FR3 Y 1.1% 2.0%

p < 0.01 ¹Previously published hotspot (Dorner et al., 1997) also in VH4signature ²As defined by Kabat ³Nucleotide position 1 is within a DWmotif, but not nucleotide positions 2 and 3.

TABLE 6 MSCSF VH4 R Mutation Frequencies at Codon Hot Spots 4-044-30^(a) 4-31 4-34 4-39 4-59 4-61 4-b Total^(d) % total VH4   2%  21%  5%   5%  18%  31%  16%   2% 1184 mutations^(b) % total VH4   6%  18%  5%   6%  24%  28%   9%   4% 145 use 30  3.8%^(e)  0.8%^(e)  1.5%^(e) 4.6%^(e)  2.3%^(e)  1.9%^(e)  1.1%^(e)  0.0%^(e)

1.9%^(d) 31B NA^(c) 3.7% 1.6% NA^(c) 7.5% NA^(c) 4.9% NA^(c)

3.5%^(d) 40 0.0% 3.3% 3.2% 6.2% 2.3% 1.9% 0.0% 0.0%

2.6%^(d) 52 3.8% 2.1% 4.8% 3.1% 2.8% 2.2% 3.8% 3.8%

2.7%^(d) 56 3.8% 6.2% 4.8% 3.1% 5.6% 2.8% 4.9% 7.7%

5.4%^(d) 57 3.8% 1.2% 0.0% 6.2% 2.3% 1.1% 2.2% 3.8%

2.0%^(d) 60 3.8% 2.5% 4.8% 0.0% 1.9% 1.4% 3.2% 0.0%

2.4%^(d) 68 0.0% 2.5% 1.6% 1.5% 0.0% 0.8% 0.5% 0.0%

1.2%^(d) 69 0.0% 0.8% 0.0% 3.1% 2.3% 3.6% 0.5% 3.8%

2.0%^(d) 81 11.5%  4.9% 6.3% 4.6% 5.6% 2.2% 4.3% 7.7%

4.6%^(d) 89 3.8% 0.0% 3.2% 4.6% 1.9% 1.9% 3.2% 7.7%

2.0%^(d) Total^(f) 34.6%  28.0%  31.7%  36.9%  34.6%  19.9%  28.6% 34.6%  27.8%  ^(a)4-30 includes all sub-genes ^(b)Percent of total Rmutations in each gene ^(c)Gene does not contain codon 31B ^(d)OverallVH4 mutational frequency (see Table 7) ^(e)Percent of total mutationsfound in this gene are at this location ^(f)Percent of total R mutationsof the gene represented in footprint

TABLE 7 MS Patient Summary¹ MS02- MS02- M125^(2,3) M199 M217 M354^(2,3)M368^(2,3) M376³ M484^(2,3) M522³ M584³ M875^(2,3) M887 19^(4,5)24^(4,5) Type of MS RR RR RR RR RR RR PP RR RR RR RR PP SP Time since MS<1  4 18 <1 15 20 3  3  1 13 3 20 diagnosis year months years year yearsyears months years month years years years Age/Sex 32/F 26/F 45/F 44/F41/F 56/F 46/F 35/F 44/F 35/F 50/F 46/F 39/F Exacerbation ONparesthesias dystonia TM TM ON myelitis TM TM ON ON NR NR History MRIFindings GD+ GD+ WML WML WML WML WML GD+ GD+ GD+, WML WML WML WML WMLClonal Expansion Yes Yes n.d. Yes Yes Yes Yes Yes Yes Yes No Yes YesOligoclonal No n.d. n.d. Yes Yes No Yes Yes n.d. n.d. No Yes Yes BandsIg Index NL n.d. High NL High n.d. High High n.d. n.d. NL High High IgSynthesis n.d. n.d. n.d. NL High n.d. High n.d. n.d. n.d. NL High HighNo. Productive 100 CSF 19  1 6 CSF 49  8 10 CSF 71 85 21  3 25/21⁶10/66⁶ VH Sequences 76 PB 19 PB 77 PB RR, Relapsing Remitting; SP,Secondary Progressive; ON, Optic Neuritis; TM, Transverse Myelitis; GD+,gadolinium enhancing; WML, White Matter Lesions; NR, not reported; n.d.,not done; NL, normal ¹All patients had CSF white blood cell (WBC) countsin the range of 1 × 10³ to 1 × 10⁴ per mL, typical of MS patients atUTSWMC (Stuve et al., 2006) ²Patient clonal analysis previouslypublished in Monson et al. (2005) ³Patient repertoire or mutationalanalysis previously published in Harp et al. (2007) ⁴Patient data notused in mutational signature analysis ⁵Patient repertoire and clonalanalysis previously published in Ritchie et al. (2004) ⁶CD19⁺/CD138⁺CSF-derived B cells

TABLE 8 OND Patient Summary¹ CIS 132⁴ CIS 429² CIS03-01⁵ OND 341⁴ OND758⁴ Sjögren's⁶ SLE⁷ HC OND CIS CIS CIS OIND NIND NA NA NA subcategoryAge/Sex 24/F 62/M 25/F 70/M 45/F 76/F 54/M 26/M; 45/M Presentation orDiplopia Optic Not Ataxia, PS HA NA NA NA Diagnosis at Neuritis reportedtime of sampling MRI Findings GD+ GD+ WML No Lesions WML ND ND ND ClonalNo Yes Yes No Yes Yes Yes No Expansion No. Productive 19 57 24/67 32 19107 41 314 VH Sequences Abbreviations: OND; Other Neurological Disease,CIS; Clinically Isolated Syndrome, NIND; non-inflammatory neurologicaldisease, OIND; Other Inflammatory Neurological Disease, HA; Headache,PS; Paraneoplastic Syndrome, GD+; Gadolinium Enhancing, WML; WhiteMatter Lesions ¹All patients had CSF white blood cell (WBC) countstypical of OND controls at UTSWMC (Stuve et al., 2006) ²This patientconverted to CDMS according to the Poser Criteria 18 months aftersampling. ³This patient's CSF analysis was negative for oligoclonalbands, and normal for Ig synthesis and rate. ⁴Patient repertoire ormutational analysis previously published in Harp et al. (2007) ⁵Thispatient converted to CDMS subsequent to this episode, and was publishedin Ritchie et al. (2004) ⁶This patient's repertoire analysis previouslypublished in Hansen et al. (2003) ⁷This patient's repertoire analysispreviously published in Hansen et al. (2000); Dorner et al. (1999)⁸CD19⁺/CD138⁺ CSF-derived B cells

TABLE 9 VH Family Usage Statistics for FIG. 1A Different DifferentDifferent Different from from from from % HCPB mHCPB MSPB MSCSF HCPB VH115.5% X N N Y n = 349 VH2 2.0% X N N N VH3 55.0% X N N Y VH4 21.8% X N NY VH567 5.7% X N N N mHCPB VH1 10.7% N X Y Y n = 205 VH2 2.0% N X N NVH3 61.5% N X N Y VH4 16.6% N X N Y VH567 9.3% N X Y Y MSPB VH1 19.9% NY X N n = 226 VH2 1.3% N N X N VH3 56.6% N N X Y VH4 17.7% N N X Y VH5674.4% N Y X N mMSPB VH1 24.2% N N N N n = 62 VH2 1.6% N N N N VH3 45.2% NY N Y VH4 22.6% N N N Y VH567 6.5% N N N N X = Not applicable;comaparing to self N = p > 0.05 Y = p ≦ 0.05

TABLE 10 VH Family Usage Statistics for FIG. 1B Different DifferentDifferent Different from from from from % HCPB mHCPB MSPB MSCSF MSCSFVH1 25.2% Y Y N X n = 405 VH2 3.2% N N N X VH3 31.4% Y Y Y X VH4 35.8% YY Y X VH567 4.4% N Y N X mMSCSF VH1 24.9% Y Y N N n = 283 VH2 1.8% N N NN VH3 31.9% Y Y Y N VH4 38.1% Y Y Y N VH567 3.3% N Y N N MSCSF VH1 23.1%Y Y N N Normal- VH2 1.7% N N N N ized VH3 32.9% Y Y Y N n = 286 VH436.7% Y Y Y N VH567 5.6% N N N N MSCSF VH1 2.3% Y N Y Y CD138 VH2 6.9% YY Y N n = 87 VH3 24.1% Y Y Y N VH4 66.7% Y Y Y Y VH567 0.0% Y Y Y Y X =Not applicable; comparing to self; N = p > 0.05; Y = p ≦ 0.05

TABLE 11 VH Family Usage Statistics for FIG. 1C Different DifferentDifferent Different from from from from % HCPB mHCPB MSPB MSCSF CISCSFVH1 2.0% Y Y Y Y n = 100 VH2 2.0% N N N N VH3 72.0% Y N Y Y VH4 22.0% NN N Y VH567 2.0% N Y N N mCISCSF VH1 0.0% Y Y Y Y n = 87 VH2 0.0% N N NN VH3 74.5% Y N Y Y VH4 23.6% N N N N VH567 1.8% N N N N CISCSF VH1 0.0%Y Y Y Y CD138 VH2 5.3% N N Y N n = 76 VH3 52.6% N N N Y VH4 42.1% Y Y YN VH567 0.0% Y Y N N N = p > 0.05; Y = p ≦ 0.05

TABLE 12 VH Family Usage Statistics for FIG. 1D Different DifferentDifferent Different from from from from % HCPB mHCPB MSPB MSCSF ONDCSFVH1 17.6% N N N N n = 51 VH2 3.9% N N N N VH3 52.9% N N N Y VH4 17.6% NN N Y VH567 7.8% N N N N Sjogren's VH1 24.3% Y Y N N Parotid VH2 4.7% NN N N n = 107 VH3 41.1% Y Y Y N VH4 19.6% N N N Y VH567 N N Y Y 10.3%SLEPB VH1 2.4% Y N Y Y n = 41 VH2 7.3% Y N Y N VH3 82.9% Y Y Y Y VH47.3% Y N N Y VH567 0.0% N Y N N N = p > 0.05; Y = p ≦ 0.05

TABLE 13 J Segment Usage Statistics for FIG. 2 Different DifferentDifferent Different from from from from % HCPB mHCPB MSPB MSCSF HCPB JH11.3% X N N N VH4 JH2 5.1% X N N N n = 78 JH3 7.7% X N N N JH4 57.7% X NY Y JH5 11.5% X N N Y JH6 16.7% X N Y N mHCPB JH1 0.0% N X NA N VH4 JH20.0% N X NA N n = 35 JH3 11.4% N X N N JH4 57.1% N X N N JH5 20.0% N X NN JH6 11.4% N X Y N MSPB JH1 0.0% N NA X N VH4 JH2 0.0% N NA X N n = 40JH3 10.0% N N X N JH4 35.0% Y N X N JH5 15.0% N N X N JH6 40.0% Y Y X YmMSPB JH1 0.0% N NA NA N VH4 JH2 0.0% N NA NA N n = 17 JH3 11.8% N N N NJH4 29.4% Y N N N JH5 0.0% N Y N Y JH6 58.8% Y Y N Y MSCSF JH1 4.9% N NN X VH4 JH2 6.3% N N N X n = 144 JH3 11.1% N N N X JH4 40.3% Y N N X JH522.9% Y N N X JH6 14.6% N N Y X mMSCF JH1 6.0% N N N N VH4 JH2 6.9% N NN N n = 116 JH3 12.9% N N N N JH4 40.5% Y N N N JH5 19.8% N N N N JH613.8% N N Y N X = Not applicable; comparing to self; NA = Notapplicable; comparing 0% to 0%; N = p > 0.05; Y = p ≦ 0.05

TABLE 14 J Segment Usage Statistics for FIG. 6 Different DifferentDifferent Different from from from from % HCPB mHCPB MSPB MSCSF HCPB JH1X N N Y n = 323 0.9% JH2 X N N N 3.1% JH3 X Y Y Y 6.8% JH4 X N Y Y 56.0%JH5 X N N Y 9.6% JH6 X N Y N 23.5% mHCPB JH1 N X N N n = 195 2.6% JH2 NX N N 2.6% JH3 Y X N N 12.3% JH4 N X Y Y 55.4% JH5 N X N Y 10.8% JH6 N XY N 16.4% MSPB JH1 N N X N n = 162 1.2% JH2 N N X N 2.5% JH3 Y N X N17.3% JH4 Y Y X Y 33.3% JH5 N N X Y 10.5% JH6 Y Y X Y 35.2% mMSPB JH1 NN N N n = 43 2.3% JH2 N N N N 0.0% JH3 N N N N 11.6% JH4 Y Y N N 30.2%JH5 N N N N 9.3% JH6 Y Y N Y 46.5% MSCSF JH1 Y N N X n = 405 4.2% JH2 NN N X 4.0% JH3 Y N N X 13.1% JH4 Y Y Y X 42.5% JH5 Y Y Y X 17.5% JH6 N NY X 18.8% mMSCF JH1 Y N N N n = 296 4.1% JH2 N N N N 4.4% JH3 Y N N N13.2% JH4 Y Y N N 42.6% JH5 Y N N N 16.2% JH6 N N Y N 19.6% X = Notapplicable; comparing to self; NA = Not applicable; comparing 0% to 0%;N = p > 0.05; Y = p ≦ 0.05

TABLE 15 CDR3 Average Statistics for FIG. 3A Average Different DifferentDifferent Different Different Different amino acid from from from fromfrom from length HCPB mHCPB MSPB mMSPB MSCSF mMSCSF HCPB All n = 348 All13.1 X Y Y Y Y Y HCPB VH4 n = 76 VH4 X N N N N N 13.4 HCPB VH3 n = 192VH3 X N Y N N N 13.0 mHCPB All n = 205 All 12.4 Y X Y Y Y Y mHCPB VH4 n= 34 VH4 N X Y N N N 12.7 mHCPB VH3 VH3 N X Y N N N n = 127 12.2 MSPBAll n = 168 All 15.3 Y Y X Y Y Y MSPB VH4 n = 40 VH4 N Y X N N N 15.7MSPB VH3 n = 89 VH3 Y Y X N Y N 14.9 mMSPB All n = 45 All 15.3 Y Y Y X YY mMSPB VH4 n = 17 VH4 N N N X N N 16.2 mMSPB VH3 n = 16 VH3 N N N X N N13.5 MSCSF All n = 405 All 13.9 Y Y Y Y X Y MSCSF VH4 n = 144 VH4 N N NN X N 14.1 MSCSF VH3 n = 129 VH3 N N Y N X N 13.3 mMSCSF All All 14.0 YY Y Y Y X n = 297 mMSCSF VH4 VH4 N N N N N X n = 116 14.2 mMSCSF VH3 VH3N N N N N X n = 91 13.6 X = Not applicable; comparing to self; N = p >0.05; Y = p ≦ 0.05

TABLE 16 CDR3 Length Range Statistics for FIG. 3B Different DifferentDifferent Different Different Different from from from from from from %HCPB mHCPB MSPB mMSPB MSCSF mMSCSF HCPB  ≦9 X N Y N N N VH4 11.5% 10-14X N N N N N 54.1% 15-19 X N N N N N 29.5% ≧20 4.9% X N Y Y N N mHCP  ≦9N X Y N N N B VH4 12.5% 10-14 N X N N N N 56.3% 15-19 N X N N N N 28.1%≧20 3.1% N X Y Y N N MSPB ≦9 0.0% Y Y X NA Y Y VH4 10-14 N N X N N N39.5% 15-19 N N X N N N 36.8% ≧20 Y Y X N Y Y 23.7% mMSP ≦9 0.0% N N NAX N N BVH4 10-14 N N N X N N 37.5% 15-19 N N N X N N 25.0% ≧20 Y Y N X YY 37.5% MSCSF ≦9 N N Y N X N VH4 13.1% 10-14 N N N N X N 44.1% 15-19 N NN N X N 35.2% ≧20 7.6% N N Y Y X N mMSC ≦9 N N Y N N X SF 14.5% VH410-14 N N N N N X 40.2% 15-19 N N N N N X 36.8% ≧20 8.5% N N Y Y N X X =Not applicable; comparing to self; NA = Not applicable; comparing 0% to0%; N = p > 0.05; Y = p ≦ 0.05

TABLE 17 MF Range Statistics for FIG. 4 Different Different DifferentDifferent Different Different from from from from from from HCPB HCPBMSPB MSPB MSCSF MSCSF % All VH4 All VH4 All VH4 HCPB All 0-4 65.0% X N NN Y Y n = 347 5-14 22.3% X N N N Y Y 15-24 9.7% X N N N Y Y ≧25 2.9% X NY N N N HCPB VH4 0-4 73.0% N X N N Y Y n = 74 5-14 17.6% N X N N Y Y15-24 6.8% N X N N Y Y ≧25 2.7% N X Y N N N HCPB VH3 0-4 58.3% N Y Y N YY n = 192 5-14 25.5% N N N N Y Y 15-24 12.0% N N N N Y Y ≧25 4.2% N N YN N N MSPB All 0-4 72.1% N N X N Y Y n = 172 5-14 21.5% N N X N Y Y15-24 6.4% N N X N Y Y ≧25 0.0% Y Y X NA Y Y MSPB VH4 0-4 58.5% N N N XY Y n = 41 5-14 31.7% N N N X N N 15-24 9.8% N N N X Y Y ≧25 0.0% N N NAX N N MSPB VH3 0-4 79.3% Y N N Y Y Y n = 92 5-14 16.3% N N N Y Y Y 15-244.3% N N N N Y Y ≧25 0.0% N N NA NA Y Y MSCSF All 0-4 26.8% Y Y Y Y X Nn = 407 5-14 45.9% Y Y Y N X N 15-24 23.1% Y Y Y Y X Y ≧25 4.2% N N Y NX N MSCSF VH4 0-4 20.1% Y Y Y Y N X n = 139 5-14 41.0% Y Y Y N N X 15-2432.4% Y Y Y Y Y X ≧25 6.5% N N Y N N X MSCSF VH3 0-4 29.1% Y Y Y Y N N n= 127 5-14 52.0% Y Y Y Y N N 15-24 17.3% Y Y Y N N Y ≧25 1.6% N N N N NY X = Not applicable; comparing to self; NA = Not applicable; comparing0% to 0%; N = p > 0.05; Y = p ≦ 0.05

TABLE 18 VH4 Mutation Frequencies at Codon Hot Spots MSCSF MSCSF MSPB toHCPB MSCSF to HCPB MSPB to MSPB HCPB Codon ALL VH4 ALL VH4 VH4 VH4 VH4ALL 30 2.3% 3.8% 2.9% 1.9%

1.8% = = p < 0.005 31B 0.3% 0.5% 1.4% 3.5%

3.6% =

p < 0.001 p < 0.001 40 0.7% 1.1% 1.5% 2.6%

  0% X = p < 0.001 52 2.5% 4.9% 2.7% 2.7%

5.5%

p < 0.005 p < 0.001 p < 0.001 56 4.6% 2.8% 4.6% 5.4%

2.7%

= p < 0.001 p < 0.001 57 2.1% 1.1% 3.2% 2.0%

  0% X = p < 0.01 60 1.3% 1.1% 1.6% 2.4%

  0% X = p < 0.001 68 1.4% 2.2% 1.0% 1.2%

0.9% = = p < 0.05 69 1.2% 1.1% 1.3% 2.0%

3.6%

= p < 0.01 p < 0.01 81 0.8% 2.7% 2.5% 4.6%

3.6% = = p < 0.001 89 1.3% 1.1% 2.1% 2.0%

1.8% = = p < 0.01 ¹MF at this position greater in VH4 than ALL ²MF atthis position less in VH4 than ALL ³MF at this position is greater inMSCSF than HCPB ⁴MF at this position is less in MSCSF than HCPB

Example 3 Materials and Methods

Patient Description.

CSF was collected from 10 RRMS patients, one PPMS patient (M484), threepatients with other neurological diseases (OND341, ataxia; OND758,headache, and OND 116, chronic inflammatory demyelinatingpolyneuropathy), and two patients with one demyelinating eventsuggestive of MS (i.e., Clinically Isolated Syndrome (CIS)) at UTSouthwestern Medical Center (UTSWMC) (Harp et al., 2007; Monson et al.,2005) in accordance with the UTSWMC Institutional Review Board (IRB).CSF was collected from nine patients with CIS at University of ColoradoDenver (UCD) as previously described (Bennett et al., 2008) inaccordance with the UCD IRB. The CIS patients had a single episode ofdemyelination (optic neuritis, brainstem or spinal cord syndrome), andthe majority had multiple lesions on MRI satisfying the dissemination inspace criterion of the McDonald criteria. None of the patients hadreceived immunomodulatory agents for at least 1 month prior to lumbarpuncture. A second relapse confirming a multiple sclerosis diagnosis hadnot occurred at the time of sample acquisition, thus not fulfilling thedissemination in time criterion (McDonald et al., 2001; Polman et al.,2005). Subsequent diagnosis of definite MS was made using the revisedMcDonald criteria (Polman et al., 2005). Conversion to definite MS wasnot revealed to the antibody sequence analysis team until aftersignature score predictions had been calculated.

MS and CIS B Cell Antibody Database Generation.

At UTSWMC, antibody repertoires were generated from CD19+ CSF B cellsusing single cell PCR as previously described (Harp et al., 2007; Monsonet al., 2005). The MSCSF database consists of antibody rearrangementsfrom 373 CD19+ CSF B cells from 10 RRMS and 1 PPMS patient recruited atUTSWMC. The CISCSF database consists of antibody rearrangements from 304CD19+ CSF B cells from 10 CIS patients (ON4-8 did not have a CD19+ CSF Bcell antibody repertoire) and 228 CD138+ CSF plasma cells from 7 CISpatients (CIS132, CIS429, ON4-10 and ON3-4 did not have CD138+ CSFplasma cell antibody repertoires). To clarify, antibody repertoires fromCIS patients at UCD were generated from both single CD19+ CSF B cellsand single CD138+ CSF plasma cells (Bennett et al., 2008), whileantibody repertoires from CIS patients at UTSWMC were generated fromsingle CD19+ CSF B cells only. Since the resultant databases (CIS CD19+CSF from UTSWMC, CIS CD19+ and CIS CD138+ CSF from UCD) were similar inmutational frequency, variable heavy chain (VH) gene family usage, andheavy chain Joining segment (JH) usage, the two databases were combinedfor analysis (Table 19).

Control B Cell Antibody Database Generation.

The healthy control peripheral blood (HCPB) antibody database has beenused in multiple studies (Brezinschek et al., 1997; 1998; Dorner et al.,1997, 1998a,b,c; Farner et al., 1999; Hansen et al., 2000; Harp et al.,2007; Monson et al., 2000; 2005) and consists of 348 CD19+ or CD19+/IgM+peripheral B cells from two healthy control donors. The memory HCPBantibody database (mHCPB) consists of 205 sequences from the HCPBantibody database that contain 4 or more mutations (less than 98%homology to the germline sequence, n=123) combined with sequences from aHCPB antibody database generated from class-switched IgD-CD27+ memory Bcells (n=82) (Tian et al., 2007) (Genbank 535266-535274, 535324-535368,535381-535408, and 535416-535418). As expected, the class-switchedIgD-CD27+ memory B cell database had a higher percentage of mutatedcodons that resulted in a replacement than the mHCPB database (compare64.7% vs 70.3%, p=0.002 by χ2 test). The OND CD19+ CSF antibody databaseconsists of 65 sequences. UCD and UTSWMC cell isolation and IgHamplification was performed similarly. All sequences were reconfirmed bythe inventors' laboratory using IgBlast (those obtained from UCD andfrom GenBank) (world-wide-web at ncbi.nlm.nih.gov/igblast/), and onlycodons 24-93 were considered in the analysis.

Mutation Analyses.

Frequency of replacement mutations (RF) was calculated as the number ofreplacement mutations at each codon position divided by the total numberof replacement mutations in each VH4 sub-database and displayed as apercentage. The MSCSF database contains 373 sequences with 475replacement mutations, and the CISCSF database contains 302 CD19+ and226 CD138+ sequences with 4081 replacement mutations (2052 in CD19+ and2029 in CD138+). The HCPB database contains 348 sequences with 1086replacement mutations, and the mHCPB database contains 205 sequenceswith 1857 replacement mutations. The ONDCSF database contains 65sequences with 482 replacement mutations, and the MSPB database contains156 sequences with 392 replacement mutations. In total, 1675 sequencesand 10,373 replacement mutations were analyzed in this manner. Table 20contains VH4 sequence numbers and Table 21 legend contains number of VH4replacement mutations. Codon domains and numbers were defined by Kabat(Kabat et al., 1983), and Tomlinson in V-base(vbase.mrc-cpe.cam.ac.uk/), respectively.

Statistical Strategy for Signature Identification.

Codons included in the signature were identified using three criteria.First, the inventors identified codons that had statistically differentRF values in the MSCSFVH4 database compared to HCPBVH4 by Goodness ofFit test where the expected frequency is the RF calculated in HCPBVH4.Twenty-four codons passed this criterion. Next, codon positions that hadan RF in both the MSCSFVH4 and HCPBVH4 databases that was less than theaverage+2 S.D. of the memory HCPBVH4 subdatabase were excluded. Thus,since the average±S.D. RF of the memory HCPBVH4 database was 0.68±0.59,any individual codon RF less than 1.86 in both databases was excluded.Fourteen codons passed this additional criterion. Eight of these 14codons (31B, 32, 40, 56, 57, 60, 81, and 89) were defined as “hot” sincethe RF at that codon position within the MSCSFVH4 database wasstatistically higher compared to the HCPBVH4 database. Six of these 14codons (30, 43, 52, 77, 82 and 82a) were defined as “cold” since the RFat that codon position within the MSCSFVH4 database was statisticallyless compared to the HCPBVH4 database. Two of the 6 “cold” codons (52and 82a) were excluded because the RF value in the MSCSFVH4 database atthat codon position was significantly higher than 1.86 (the average+2S.D. of the memory HCPBVH4 subdatabase). The overall signatureconsequently consisted of codons 30, 31B, 32, 40, 43, 56, 57, 60, 77,81, 82, and 89. This analysis was not biased by differences in theprevalence of particular codons (31B in particular), as individual VH4gene frequencies in MSCSF were similar to HCPB by χ 194 2 test using aBonferroni corrected p-value of 0.004 (data not shown).

Statistical Computation of the Signature Score.

Signature scores were generated by calculating Z-scores for the RFvalues at the 6 codons within the signature (31B, 40, 56, 57, 81 and 89)that had the most significant difference in RF compared to HCPBVH4 ateach codon position. The Z-score formula is: (RF at codon X minus theaverage RF in HCPBVH4)/(standard deviation of the average RF inHCPBVH4). For example, the average RF in HCPBVH4 within the 6 signaturecodons was 1.6±0.9 and so an RF of 4.4 at codon 31B would be assigned ascore of 3.1 (Z-score=(4.4−1.6)/0.9). Individual Z-scores at each of the6 codon positions were then added to generate the composite signatureZ-score. The average composite signature score in the MSCSFVH4 databasewas 10.9±2.0 and so any signature score of an individual CIS patientabove 6.8 (average−2 S.D.) was predicted to convert to CDMS. Of note,both the ONDCSFVH4 signature score (at 4.5), and the MSPBVH4 score (at2.0) were below the threshold for MS conversion. CD19+ CSF B cell andCD138+ CSF plasma cell mutation positions both contributed to each CISpatient's signature score, while the MSCSFVH4 signature scores were onlycomposed of CD19 214+CSF B cells. VH4 structure. A human VH4-30.4antibody structure described in (Guddat et al., 1993) was obtained fromthe Protein Data Bank (world-wide-web rcsb.org) under the identificationmoniker 1MCO, and adapted using the RasMol program(mc2.cchem.berkely.edu/Rasmol/) to highlight codons within thedesignated signature of the heavy chain variable region.

Example 4 Results

The 51 antibody heavy chain variable genes are subdivided into 7different families (Cook and Tomlinson, 1995; world-wide-web atncbi.nlm.nih.gov/igblast/), and it has been well-established thatperipheral blood B cells from healthy donors utilize VH antibody genesmost often from the VH3 family (“HCPB” in Table 20 and (Brezinschek etal., 1995; Brezinschek et al., 1997; Huang et al., 1992; Kraj et al.,1997; Wardemann et al., 2003; Yurasov et al., 2005)). In contrast, ithas been reported by us and others that B cells in the CSF of MSpatients often utilize VH4 antibody genes more frequently than those inthe VH3 family (“MSCSF” in Table 20 and (Baranzini et al., 1999; Colomboet al., 2000; Harp et al., 2007; Monson et al., 2005; Owens et al.,1998; 2003; 2007; Qin et al., 1998; Ritchie et al., 2004)). The CISCSFantibody database consisting of CD19+ B cells only had a similarfrequency of B cells that utilize VH4 family genes in comparison to HCPB(26.2% vs. 21.8%, p=0.20 by χ2 test) (Table 20); in contrast, whenCD138+ plasma cells were included, the CISCSF had a higher frequency ofB cells that utilize VH4 family genes in comparison to HCPB (35.0% vs.21.8%, p=0.00001 by χ2 test) (Table 20). Some individual CIS patient CSFB cell antibody repertoires were enriched for B cells utilizing VH4family genes in comparison to the random expected frequency, as reportedpreviously (Bennett et al., 2008). CSF-derived B cell antibodyrepertoires from patients with Other Neurological Diseases (OND) werenot enriched for VH4-expressing CSF B cells in comparison to HCPB (23.1%vs. 21.8%, p=0.83 by χ2 test) or mHCPB (23.1% vs. 16.6%, p=0.24 by χ2test), indicating that VH4 over-expression in the CSF of MS patients wasnot due to bias in the ability of VH4 expressing B cells to enter theCNS.

Identification of Codons within MSCSF that are Enriched for ReplacementMutations.

Since VH4 expressing B cells are enriched in the CSF of MS patients, wehypothesized that mutational analysis would reveal a pattern (i.e.,“signature”) of antibody gene replacement mutations that is unique toVH4 expressing B cells from the CNS of MS patients in comparison toHCPB. In order to test this hypothesis, the percentage of replacementmutations (RF) at each codon within the VH4 subdatabase extracted fromthe parent database (MSCSFVH4) was determined and compared to the RF ateach codon position within the VH4 subdatabase extracted from the parentHCPB database (HCPBVH4). Replacement frequencies were used so that onlythose mutations resulting in an amino acid change would be considered.In addition, codon amino acid replacement can result from 1, 2, or 3nucleotide changes within the codon, and so replacement frequencieslimit bias based on the number of nucleotides in a codon that aremutated to generate a replacement. Hot spots were defined as those codonpositions within MSCSFVH4 with a statistically higher RF at a particularcodon position in comparison to HCPBVH4 (Table 21). Using this approach,8 codon positions (31B, 32, 40, 56, 57, 60, 81, and 89) were identifiedthat have a total RF value in MSCSFVH4 (25.0%) that was statisticallyhigher than in HCPBVH4 (12.6%) (p=0.001 by χ2 test). Cold spots weredefined as those codon positions within MSCSFVH4 with a statisticallylower RF at a particular codon position in comparison to HCPBVH4 (Table21). Four codons (30, 43, 77 and 82) were identified as cold spots thathave a total RF value in MSCSFVH4 (5.1%) that was statistically lessthan in HCPBVH4 (8.5%) (p=0.001 by χ2 test).

Individual MS patient RFs within the 8 hot spot codons of the signatureranged from 22.5 to 34.1% (data not shown), indicating that someindividual patient MSCSF repertoires had a greater enrichment ofreplacements at these 8 codon positions than others. Also, thevariability of RF values within the 8 hot spot codons of the signaturein individual VH4 genes in MSCSFVH4 ranged from 14.5 to 36% data notshown), indicating that some individual VH4 genes had a greaterenrichment of replacements at these 8 hot spot codon positions thanothers. Previous analysis had identified codon 56 as a replacementmutation hotspot in HCPB (Dorner et al., 1997; Dorner et al., 1998a),which intensified as a hot spot in MSCSF_(VH4) since a significantlygreater percentage of replacement mutations were found in MSCSF_(VH4) atcodon 56 compared to HCPB_(VH4). Of note, there was a 7.0-fold increasein replacement accumulation at codon 31B in the MSCSF_(VH4) database incomparison to HCPB_(VH4) that is likely due to the use of this codon byonly a subset of VH4 genes (4-30, 4-31, 4-39 and 4-61).

When the analysis was restricted to those B cells expressing VH4 genesthat contain codon 31B, there was a 3.1-fold increase in RF of theMSCSF_(VH4) database compared to HCPB_(VH4) (pb 0.001). An example of asignature-enriched VH4 antibody gene rearrangement from a CSF-derived Bcell of an MS patient is provided in FIG. 8. Of note, 5 of the 8 hotspot codons of the signature retained higher RF values in MSCSF comparedto the memory HCPB database (31B, 40, 56, 57, and 60), emphasizing thatthe signature does not simply reflect enrichment of memory B cells inthe CSF.

Potency of Signature Score to Predict Development of Clinically DefiniteMS.

The inventors reasoned that prevalence of the signature would allow themto identify patients at risk to develop MS who subsequently convert toCDMS. Current criteria for diagnosis of MS requires dissemination oflesions both in time and space (Barkhof et al., 1997; Polman et al.,2005; Tintore et al., 2000). When MRI lesions alone are not sufficientto confirm diagnosis, CSF abnormalities can be used to meet the criteriaof dissemination in space (Polman et al., 2005; Siritho and Freedman,2009). Risk of conversion to clinically definite MS in patients who havehad a single demyelinating event is 50-90% if the patient has anabnormal MRI (Beck et al., 2003; Brex et al., 2002; Cole et al., 1998;O'Riordan et al., 1998; Soderstrom et al., 1998), but of those patientswith a normal MRI, up to 29% had oligoclonal bands and converted to CDMS(Cole et al., 1998).

In order to test whether signature prevalence could predict conversionto MS, the inventors generated CSF B cell repertoires from patients whohad one demyelinating event that placed them “at risk” to develop MS.Such patients are typically diagnosed with CIS. CD19+ B-cell and CD138plasma cell repertoires from the CSF of two CIS patients at UTSWMC andnine patients at UCHSC were generated and analyzed for RF values withinthe 6 codons of the signature defined in the MSCSF_(VH4) database thathad the most significant difference in RF compared to HCPB_(VH4) at eachcodon position (codons 31B, 40, 56, 57, 81 and 89). RF values werecombined using a signature score that accounts for RF variance asdescribed in Materials and methods. The average signature score in theMSCSF_(VH4) database was 10.9±2.0 (range 7.6-11.9), and so anyindividual CIS patient score that was 6.8 (average signature score−2S.D.) or higher was predicted to develop MS (FIG. 9 and Table 22).Notably, the signature score from a pool of VH4 expressing CSF B cellsof 3 OND patients was 4.5, and the signature score from a pool of VH4expressing peripheral blood B cells of 3 CDMS patients was 2.0, and thusdid not reach the 6.8 signature score threshold. Also, signature scoresbased on CD19+ B cell sequences only (in the patients where this waspossible) did not change predictions based on signature score. This wasexpected since there is significant overlap in the antibody generepertoires of CD19+ B cells and CD138+ plasma cells from the CSF of thesame patients (Martin Mdel and Monson, 2007; Ritchie et al., 2004),suggesting that the memory B cell pool present in the CSF is thereservoir for differentiation of plasma cells in the CSF.

As indicated in Table 22, prediction of conversion to CDMS using theantibody gene signature score was accurate in 8 of 8 CIS patients thatconverted to CDMS. Lack of signature prevalence also accuratelypredicted that 2 of 2 patients who had recently experienced a firstdemyelinating event (ON3-1 and ON4-10) would not develop CDMS, andindeed, have not developed CDMS up to 2 years after initial sampling.One additional patient who had recently experienced a firstdemyelinating event (ON3-4) had a high signature score (11.3), but hadnot converted to CDMS at the 2-year follow-up. The antibody genesignature yielded a sensitivity of 100%, specificity of 67%, positivepredictive value of 89%, negative predictive value of 100%, and accuracyof 91%, as defined by others applying the McDonald Criteria to identifyCIS patients that would convert to MS (Dalton et al., 2002). Mostpatients in this cohort converted to CDMS within 3-6 months ofrepertoire sampling, although in the case of CIS 132, conversion to CDMSwas not confirmed until 17 months after antibody repertoire sampling(Table 22). MRI, OCB and VH4/VH2 bias are also useful in assessingprobability of MS conversion (Bennett et al., 2008; Freedman et al.,2005; Frohman et al., 2003; Korteweg et al., 2006; Paolino et al., 1996;Soderstrom et al., 1998), but were not considered in calculating thesignature score.

Example 5 Discussion

The intense somatic hypermutation accumulation in MSCSF_(VH4) enabled usto identify a unique antibody gene signature-enriched for replacementaccumulation at codons 31B, 32, 40, 56, 57, 60, 81 and 89 that was notobserved in HCPB_(VH4) or ONDCSF_(VH4). Of note, any residual effect ofnaïve B cells on the RF calculation was minimized by tabulating onlythose sequences with mutations resulting in amino acid replacements.This approach minimized bias in the signature that may have reflectedenrichment of mutation accumulation in CNS derived B cells (which aremostly memory and thus have high mutation rates) compared to peripheralB cells (which are mostly naïve and thus have low mutation rates). Inaddition, 5 of the 8 hot codons of the signature retain higher RF valuesin the MSCSF database compared to the memory HCPB database. Finally, ifsignature score reflected enrichment of mutation accumulation due to therepertoire's high memory representation, then all signature scores fromthe CISCSF antibody repertoires should have been high since they wereall heavily enriched for memory B cells. This was not the case, sinceCISCSF repertoires ON3-1 and ON4-10, despite being heavily enriched formemory B cells (with mutation frequencies of 5.2% and 6.7%,respectively), had signature scores below the threshold of 6.8 (ON3-1score=6.4, ON4-10 score=2.2).

It was compelling to investigate whether the antibody gene signature maybe of value to identify CIS patients who would subsequently develop MS,since early and accurate diagnosis of MS is of tantamount importance inclinical care (Stuve et al., 2008). Signature prevalence could be usedto identify patients who would be diagnosed with CDMS within 3-18 monthsof experiencing their first demyelinating event. Of note, patient ON3-4had a signature score that indicated this patient would convert to CDMS(score=11.3), but did not demonstrate a lesion load by MRI, banding byOCB, or VH4/VH2 bias, and had not developed CDMS up to 2 years aftersampling was performed (Table 22). It will be interesting to determinewhether this patient is diagnosed with CDMS over time. It is alsoimportant to note that the majority of patients in this cohort alreadyhad evidence of MS risk as indicated by positive MRI and OCB. PatientON4-7, however, did not present with brain lesions by MRI, but had asignature score that indicated this patient would be diagnosed with CDMS(score=10.2). Indeed, this patient did convert to CDMS within 5 monthsof CSF B cell antibody repertoire sampling, and provides a reasonableexample of how signature prevalence may predict CDMS diagnosis inpatients that either do not present with brain lesions by MRI, or whoare not evaluated by MRI at this stage of their disease. It will beinteresting to determine whether the combination of MRI and signatureprevalence would be useful in predicting MS conversion. Signatureprevalence may also provide an evaluation mechanism to identify the mostappropriate patient candidates to receive B cell depletion therapies,for example. Certainly this is a priority since a recent investigationdemonstrated significant efficacy of Rituxan in RRMS patients (Hauser etal., 2008).

Given the urgency for the early identification of MS and the rapidinitiation of disease modifying therapy, presentation of a molecularsignature in the CSF B cells of CIS patients who develop MS may providea unique tool for identifying at risk individuals. However, wideimplementation of the current form of this approach would be problematicsince the AGS scores presented here were generated using a specializedtechnique that is labor intensive (single cell PCR) and requires freshCSF for sampling. Developing other approaches to generate AGS data thatmaintains accuracy, but does not require a specialized laboratory toperform, is attainable and of paramount importance.

In addition, many early MS patients have atypical clinical presentationsor unremarkable MRI scans, and patients with alternative inflammatoryconditions may mimic idiopathic demyelinating disease. In thesecircumstances, the advent of a molecular diagnostic signature wouldincrease diagnostic sensitivity and specificity. Investigating theutility of the antibody gene signature in such patients is ongoing inthe inventors' laboratory.

The presence of a mutational signature among clonally expanded VH4germline antibodies in MSCSF may be helpful in understanding diseasepathogenesis. For example, the VH4 germline mutational signature may bethe direct result of antigen targeting in the humoral immune response.Therefore, determining the antigen specificity of signature-enrichedantibodies from CSF B cells of patients with definite MS and CIS is oneof the first steps towards dissecting whether signature-enriched B cellshave the potential to participate in MS pathogenesis. Of note, 5 of the8 signature codons (31B, 32, 56, 57 and 60) the inventors identified ashaving a unique accumulation of amino acid replacements in MSCSF_(VH4)are predicted to have direct antigen contact since they reside in CDRs(FIGS. 10A-B). Dissecting the relative contribution of replacementmutations at each of these signature codons as well as those outside ofthe antigen binding region will address the impact of both codonclassifications (direct and indirect antigen binding capacity) onantigen binding affinity.

In summary, a unique signature of antibody gene replacement mutationswas identified in the MSCSF_(VH4) database that is not observed inhealthy control peripheral blood or CSF-derived VH4-expressing B cellsfrom patients with other neurological diseases. Prevalence of thesignature was accurate in identifying CIS patients that would convert toCDMS, but needs to be tested on a larger cohort of patients at both highand low risk to develop MS. Identifying the antigen specificity ofsignature-enriched CSF B cells from these patients may also reveal aunique group of antigens that are central to initiation of humoralautoimmunity in the CNS. It is likely that the MS-specific VH4 antibodygene signature provides both a new focus of investigation to furtherelucidate the role of B cells and their antibody products in MS and anew candidate molecular diagnostic tool for MS.

TABLE 19 CISCSF B Cell Repertoire Source Comparison CISCSF CISCSFCD19⁺ + Cell Source CD19⁺ CD138 p value VH1 9.9 8.9 NS VH2 5.3 7.0 NSVH3 56.0 47.5 0.02 VH4 26.5 35.0 0.01 JH1 2.7 2.5 NS JH2 3.7 2.6 NS JH311.6 11.2 NS JH4 44.9 42.9 NS JH5 15.0 14.0 NS JH6 22.3 26.8 NS % ofmutated 68.4 68.4 NS codons resulting in a replacement^(a) MF^(b) 5.35.7 <0.001 Abbreviations used in this table: CIS = clinically isolatedsyndrome; CSF = cerebrospinal fluid; RF = replacement frequence; MF =mutational frequence; NS = not significant ^(a)Number of mutated codonscausing replacement divided by the number total mutated codons(replacement and silent) ^(b)Number of mutations divided by read length(codons 31 through 93 for both calculations)

TABLE 20 Frequences of VH Family Usage^(a) Expected Frequence HCPB^(b)mHCPB^(c) MSCSF^(d) CISCSF^(e) CISCSF^(f) ONDCSF^(g) B Cell Source ByGene CD19⁺ CD19⁺ Frequency^(j) CD19⁺ IgD⁻CD27⁺ CD19⁺ CD19⁺ CD138⁺ CD19⁺VH1 21.6 15.5 10.7 27.1^(h,i) 9.9 8.9 20.0 VH2 5.9 2.0 2.0 1.6 5.3^(h)7.0 3.1 VH3 43.1 55.2 62.0 32.4^(h,i) 56.3 47.5 46.2^(i) VH4 21.6 21.816.6 34.3^(h,i) 26.2^(i) 35.0 23.1 Total Sequences 51 348 205 373 302528 65 Number of 0 2 6 11 10 11 3 Donors Abbreviations: VH, variableheavy; HCPB, healthy control peripheral blood; mHCPB, memory healthycontrol peripheral blood; MSCSF, multiple sclerosis cerebrospinal fluid;CISCSF, clinically isolated syndrome cerebrospinal fluid; ONDCSF, otherneurological disease cerebrospinal fluid. ^(a)Values provided inpercent. ^(b)The HCPB group includes CD19⁺ B cell antibody sequencesfrom healthy controls BF1 (n = 67) and BF2 (n = 281). ^(c)The mHCPBgroup includes CD19⁺ B cell antibody sequences from healthy controls BF1(n = 18) and BF2 (n = 105) with 4 or more mutations (less than 98%homology to germline) and IgD⁻CD27⁺ B cell antibody sequences fromhealthy controls HA (n = 9), HB (n = 44), HC (n = 26), and HE (n = 3)(Tian et al., 2007) ^(d)The MSCSF group includes CD19⁺ sequences from MSpatients M125 (n = 101), M199 (n = 19), M354 (n = 6), M368 (n = 49),M376 (n = 8), M484 (n = 9), M522 (n = 71), M584 (n = 85), M875 (n = 21),M217 (n = 1), and M887 (n = 3). ^(e)The CISCSF group includes CD19⁺sequences from CIS patients CIS132 (n = 19), CIS429 (n = 57), CIS3-1 (n= 24), ON3-1 (n = 23), ON3-3 (n = 39), ON3-4 (n = 28), ON3-5 (n = 35),ON4-7 (n = 17), ON4-10 (n = 31), and ON5-2 (n = 29). ^(f)In addition tothose CD19⁺ sequences, this group includes CD138+ sequences from CISpatients CIS3-1 (n = 76), ON3-1 (n = 45), ON3-3 (n = 12), ON3-5 (n =44), ON4-7 (n = 20), ON4-8 (n = 17), ON5-2 (n = 12). ^(g)The ONDCSFgroup includes CD19⁺ B cell antibody sequences from OND patients OND341(n = 32), OND758 (n = 19), and OND116 (n = 14). ^(h)Significantlydifferent from HCPB frequency ^(i)Significantly different from mHCPBfrequency ^(j)Expected frequency from (Cook and Tomlinson, 1995)

TABLE 21 Percentage of Replacement Mutations in Each Signature CodonMSCSF_(VH4) HCPB_(VH4) mHCPB_(VH4) Codon Location^(b) RF RF FoldIncrease p-value^(d) RF Fold Increase p-value^(d) 31B^(c) CDR1 3.5⁵ 0.5⁵7.0 0.001 0.8 4.4 0.001 32 CDR1 2.3 1.5 1.5 0.05 2.1 1.1 NS 40^(c) FR22.7 1.0 2.7 0.001 1.1 2.5 0.001 56^(a,c) CDR2 5.5 3.0 1.8 0.001 3.2 1.70.001 57^(c) CDR2 2.0 1.0 2.0 0.005 0.5 3.7 0.001 60 CDR2 2.4 1.5 1.60.05 1.1 2.2 0.001 81^(c) FR3 4.7 3.0 1.5 0.005 3.7 1.3 NS 89^(c) FR32.0 1.0 2.0 0.005 1.3 1.5 NS Hotspot Total^(c) 25.0 12.6 2.0 0.001 13.81.8 0.001 30 FR1 2.0 4.0 0.5 0.005 2.9 0.7 NS 43 FR2 0.9 2.0 0.5 0.0251.3 0.7 NS 77 FR3 1.5 2.5 0.6 0.05 1.6 0.9 NS 82 FR3 0.7 2.5 0.3 0.0011.6 0.5 0.05  Cold spotTotal^(c) 5.1 8.5 0.5 0.001 7.4 0.7 0.01 Abbreviations in table: CDR, complementary determining region; FR,framework; RF, replacement frequency; MSCSF, multiple sclerosiscerebrospinal fluid; HCPB, healthy control peripheral blood; mHCPB,memory HCPB; NS, not significant ^(a)Previously published replacementhotspot (Dorner et al., 1997; Dorner et al., 1998a) ^(b)As defined byKabat (Kabat et al., 1983) ^(c)“Hotspot Total” is the total RF withincodons 31B, 32, 40, 56, 57, 60, 81 and 89. “Coldspot Total” is the totalRF within codons 30, 43, 77, and 82. 199, 337 and 965 replacementsrespectively were included in this analysis for HCPB_(VH4), mHCPBVH4,and MSCSF_(VH4.) ^(d)Comparing HCPB_(VH4) or mHCPB_(VH4) to MSCSF_(VH4)RFs at each codon position using χ² goodness-of-fit where expectedfrequence is the RF calculated in HCPB_(VH4) respectively. ^(e)Codonused in the calculation of signature score

TABLE 22 CIS Patient Summary and Signature Score Predictions PredictionCD19 Based On Subject Time to MRI Brain CD19/ VH4 CD138 SignatureSignature Definite Time to MS No.^(a) LP^(b) Lesions OCB CD138^(c)bias^(d) VH4 bias^(d) Score^(e) Score MS Diagnosis^(f) CIS132 1 GD⁺ Yes19/NA No NA 12.1 CDMS Clinical 18 CIS429 1 GD⁺ Yes 56/NA No NA 15.0 CDMSClinical 3 CIS3-1 2 WML Yes 24/76 Yes Yes 15.5 CDMS MRI 3 ON3-3 4 GD⁺Yes 39/13 No No 11.3 CDMS Clinical 3 ON3-5 1.75 GD⁺ Yes 35/44 Yes Yes12.8 CDMS Clinical 2 ON4-7 3 None^(g) Yes 17/20 No Yes 10.2 CDMSClinical 5 ON4-8 1.5 WML Yes NA/18 NA Yes 9.6 CDMS Clinical 5 ON5-2 1GD⁺ Yes 29/12 Yes Yes 7.9 CDMS Clinical 3 ON3-1 10 WML Yes 23/45 No No6.4 No — NA ON4-10 1.25 WML No 31/NA No NA 2.2 No — NA ON3-4 1.5None^(g) No 28/NA No NA 11.3 CDMS — NA Abbreviations in table: CIS,Clinically Isolated Syndrome; ON, Optic Neuritis; LP, lumbar puncture,GD, gadolinium enhancing lesion positive; WML, white matter lesions byT2, OCB, oligoclonal bands; CDMS, clinically definite MS; NA, notapplicable ^(a)CIS132 and CIS429 were generated at UTSWMC; the remainingpatient CSF B cell repertoires were generated at UCD. ^(b)monhts fromfirst demyelinating event to LP ^(c)Values given are number of uniquesequeces in CD19 repertoire/CD138 repertoire; family usage can be foundin (Bennett et al., 2008; Harp et al., 2007); CIS429 was a 62 y.o. malefirst presenting with optic neuritis and the repertoire had 2% VH1usage, 0% VH2, 77% VH3 and 19% VH4. ^(d)Bias was consideredsignificantly different from random frequence (Cook and Tomlinson, 1995)or expected frequence in HCPB (Brezinschek et al., 1997; Brezinschek etal., 1998; Dorner et al., 1997; Dorner etr al., 1998a, Dorner et al.,1998b; Dorner et al., 1998c; Farner et al., 1999; Hansen et al., 2000;Monson et al., 2000). VH2 bias was also observed in CD19 repertoiresfrom ON4-7, and CD138 repertoires from ON3-3, ON3-5 and ON4-7.^(e)Signature score was calculated as outlined in materials and methods,and uses both CD19 and CD138 sequences. The average score among MSpatients is 10.9 ± 2.0. ^(f)Months from first demyelinating event to MSdiagnosis. ^(g)One spinal cord lesion was observed by T2 weighted MRI.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

V. REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

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What is claimed is:
 1. A method for identifying a human subject havingor at risk of developing multiple sclerosis (MS) comprising assessingthe VH4 structure of a VH4-expressing B-cell from said subject, whereinthe presence of a codon signature associated with MS identifies saidsubject as having or at risk of developing MS.
 2. The method of claim 1,wherein said codon signature comprises a mutation at codon 31B, 56and/or
 81. 3. The method of claim 2, wherein said codon signaturecomprises mutations at each of 31B, 56 and
 81. 4. The method of claim 2or 3, wherein said codon signature further comprises mutations at one ormore of codons 32, 40, 57, 60 and
 89. 5. The method of claim 4, whereinsaid codon signature comprises mutations at each of codons 31B, 32, 40,56, 57, 60, 81 and
 89. 6. The method of claim 1, wherein said codonsignature comprises a mutation at codons 31B, 40, 56, 57, 81 and/or 89.7. The method of claim 6, wherein said codon signature comprises amutation at each of codons 31B, 40, 56, 57, 81 and
 89. 8. The method ofclaim 1, further comprising assessing one or more traditional MS riskfactors.
 9. The method claim 1, wherein assessing comprises sequencing.10. The method of claim 1, wherein assessing comprises PCR.
 11. Themethod of claim 1, wherein said B-cell is obtained from cerebrospinalfluid (CSF).
 12. The method of claim 11, further comprising assessing Jchain usage, J chain length and/or CDR3 length.
 13. The method of claim1, wherein said B-cell is obtained from peripheral blood.
 14. The methodof claim 13, further comprising assessing J chain usage, J chain lengthand/or CDR3 length.
 15. The method of claim 1, further comprising makinga treatment decision based on the presence of said codon signature. 16.A method of screening for an agent useful in treating multiple sclerosis(MS) comprising: (a) providing an antibody produced by a VH4-expressingB-cell, said antibody comprising mutations at three or more codonsselected from the group consisting of 31B, 32, 40, 56, 57, 60, 81 and89; (b) contacting said antibody with a candidate ligand; and (c)assessing binding of said candidate ligand to said antibody, whereinbinding of said candidate ligand to said antibody identifies saidcandidate ligand as useful in treating MS.
 17. The method of claim 16,wherein said candidate ligand is a peptide or a peptoid.
 18. A method oftreating a subject having or at risk of developing MS comprisingadministering to said subject a ligand that binds to an antibody VH-4antibody comprising mutations at three or more codons selected from thegroup consisting of 31B, 32, 40, 56, 57, 60, 81 and
 89. 19. The methodof claim 18, wherein said ligand is a peptide or a peptoid.
 20. Themethod of claim 18, wherein said ligand is linked to a toxin or B-cellantagonist.